©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Membrane-bound Phospholipase D by Protein Kinase C in HL60 Cells
SYNERGISTIC ACTION OF SMALL GTP-BINDING PROTEIN RhoA (*)

(Received for publication, August 28, 1995; and in revised form, November 10, 1995)

Kenji Ohguchi Yoshiko Banno Shigeru Nakashima Yoshinori Nozawa (§)

From the Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In HL60 cells, the membrane-bound phospholipase D (PLD) was stimulated by 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of the cytosolic fraction from HL60 cells or rat brain. The cytosolic factor for this PMA-induced PLD activation was subjected to purification from rat brain by sequential chromatographies. The PLD stimulating activity was found in protein kinase C (PKC) fraction containing alpha, betaI, betaII, and isozymes. PKC isozymes were further separated by hydroxylapatite chromatography. PKCalpha and -beta, but not , isozymes were found to activate membrane-bound PLD. PKCalpha was much more effective than PKCbeta for PLD activation. Millimolar concentrations of MgATP were required for the PKC-mediated PLD activation in HL60 membranes. MgATP is utilized to maintain the levels of phosphatidylinositol 4,5-bisphosphate (PIP(2)) under these assay conditions. The PKC-mediated PLD activation was completely inhibited by neomycin, a high affinity ligand for PIP(2), and this suppression was recovered by the addition of exogenous PIP(2). Thus, these results suggest that PIP(2) is supposed to play a key role in PKC-mediated PLD activity in HL60 membranes. Furthermore, PKCalpha-mediated PLD activation was potentiated by the addition of recombinant RhoA protein in the presence of guanosine 5`-O-(3-thiotriphosphate) (GTPS). The results obtained here indicate that PKCalpha and RhoA (GTP form) exert a synergistic action in the membrane-bound PLD activation in HL60 cells.


INTRODUCTION

Phospholipase D (PLD) (^1)has been recognized to play an important role in signal transduction of many types of cells. PLD hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline(1) . PA and its dephosphorylated product diacylglycerol are important second messengers. PLD is activated in a variety cells in response to receptor agonists, phorbol ester and Ca ionophore(2) . Recently, it has been pointed out that several factors are required for activation of PLD. In reconstitution experiments, activation of membrane-bound PLD induced by phorbol 12-myristate 13-acetate (PMA) or nonhydrolyzable guanine nucleotide (GTPS) was observed only when cytosol and membranes were present(3) . Similar findings were obtained in permeabilized cell preparations in which leakage of cytosolic components resulted in reduction of PLD activity(4, 5) . These results imply that cytosolic factors for PLD activation are involved in protein kinase C (PKC) and GTP-binding proteins.

PKC has been reported to be implicated in PLD activation in various cell types. Evidence that PKC up-regulates PLD activity is supported by the observations that PKC inhibitors or PKC down-regulation by long-term exposure to PMA prevents the increase of PLD activity(2) . Recently, a role for specific PKC isozymes in the regulation of PLD is presented by the studies overexpressing PKCalpha or -beta isozymes in cells. Overexpression of PKCbetaI enhances PMA-induced PLD activity in rat fibroblasts(6) . Overexpression of PKCalpha in Swiss-3T3 fibroblasts (7) does not induce an acute stimulation of PLD by PMA, but rather it plays a role in the expression of PLD enzyme. Furthermore, in membranes isolated from CCL39 fibroblasts(8) , only PKCalpha and -beta are capable of activating PLD. However, exact molecular interactions between PLD and PKC have not yet been studied.

On the other hand, stimulation of PLD activity by GTPS in permeabilized cells and cell lysates indicates its regulation by GTP-binding proteins(2) . Recent studies have demonstrated the implication of two small GTP-binding proteins, ADP-ribosylation factor (ARF) (9, 10) and Rho (11, 12) in the regulation of PLD activity in several types of cells. Furthermore, in some types of cells (13, 14, 15, 16, 17, 18, 19, 20) , PLD activation induced by both GTPS and PMA was greatly enhanced, compared with that caused by either stimulant alone. These findings suggest that PKC may play an important role in positively modulating GTP-binding protein-mediated PLD activity. However, the precise relationship between GTP-binding proteins and PKC has not yet been disclosed. Our recent study (20) has demonstrated that the partially purified PKC fraction from rat brain cytosol showed a synergistic stimulation of PLD activity of HL60 membranes by PMA and GTPS. Moreover, this synergistic activation of the membrane-bound PLD was prevented by pretreatment with Rho GDP dissociation inhibitor (RhoGDI), suggesting a potential role of RhoA in the PKC-mediated PLD activation.

The present study was designed to gain more insight into the mechanisms underlying the PMA-induced PLD activation in HL60 membranes. A cytosolic factor reconstituting PMA-induced PLD activity was resolved as PKC fraction from rat brain. Among PKCalpha, -beta, and - isozymes, PKCalpha was the most effective in activating membrane-bound PLD. PKCalpha-mediated PLD activation was synergistically stimulated by RhoA in the presence of GTPS. Furthermore, MgATP and phosphatidylinositol 4,5-bisphosphate (PIP(2)) were necessary for the membrane-bound PLD activation by the partially purified PKC fraction which was free from small GTP-binding proteins of brain cytosol.


EXPERIMENTAL PROCEDURES

Materials

HL60 human promyelocytic leukemic cell line was kindly supplied by Dr. T. Okazaki (Osaka Dental University). [^3H]Oleic acid and [palmitoyl-^3H]DPPC were obtained from DuPont NEN, myo-[^3H] inositol and [-P]ATP were from Amersham. PMA, 4alpha-PDD and PIP(2) were from Sigma. GTPS was from Boehringer Mannheim. Antibodies against PKC isozymes (alpha, betaI, betaII, , , , , , and ), small GTP-binding proteins (RhoA, Rac1/Rac2, Cdc42Hs) and RhoGDI were from Santa Cruz Biotechnology. Silica Gel 60 (LK6D) plates were from Whatman. Mono Q and Superose 12 columns were from Pharmacia Biotech Inc. Hydroxylapatite was from Mitsui-Tohatsu Chemicals. Protein determination reagents were from Bio-Rad. Recombinant RhoA and GST-RhoGDI were kindly supplied by Dr. Y. Takai (Osaka University).

Cell Culture and Cell Labeling

HL60 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were grown at a cell density of 0.2-1.0 times 10^6 cells/ml in a humidified atmosphere containing 5% CO(2) at 37 °C. For assay of PLD activity, cells were labeled with [^3H]oleic acid (0.5 µCi/ml) for 12-15 h. Under these conditions, 60-65% of total radioactivity incorporated into cells was found in the phosphatidylcholine (PC) fraction. For the analysis of phosphoinositides, cells were labeled with myo-[^3H]inositol (2 µCi/ml) in inositol-free alpha-minimum essential medium supplemented with 2 mM glutamine and 3.5 mg/ml bovine serum albumin for 24 h.

Preparation of Membranes and Cytosol Fractions from HL60 Cells

Membranes and cytosol fractions were prepared by the method described by Olson et al.(3) , with minor modifications. The labeled cells were washed twice with buffer A (25 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 5 mM MgCl(2), 0.5 mM MgATP, 1 mM EGTA, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml leupeptin) and resuspended in buffer A. Cells were then disrupted by N(2) cavitation (600 p.s.i. at 4 °C for 30 min). After unbroken cells were removed by centrifugation at 900 times g for 5 min, membrane and cytosol fractions were separated by centrifugation at 100,000 times g for 60 min. Membranes were washed once and resuspended in buffer A. Membranes were used within 12 h after isolation. Cytosol proteins were concentrated using Centricon 10 (Amicon) and stored at -80 °C until use.

Assay of PLD Activity in HL60 Membranes

[^3H]Oleic acid-labeled HL60 membranes (50 µg of protein/assay) and crude cytosol or purified PKC fractions were incubated in buffer A containing CaCl(2) to give a final free Ca concentration of 1 µM (total 0.1 ml) and were stimulated with 100 nM PMA and/or 10 µM GTPS at 37 °C for 15 min in the presence of butanol (0.3%, v/v). Reactions were terminated by the addition of chloroform/methanol (1:2, v/v). Lipids were extracted according to the method of Bligh and Dyer (21) and separated on Silica Gel 60 TLC plates in a solvent system of the upper phase of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10, v/v) as described previously(22) . The plates were exposed to iodine vapor, and [^3H]PBut was identified by comigration with PBut standard which was prepared by the partially purified cabbage PLD(23) . The spots scraped off the plates were mixed with scintillation mixture, and the radioactivity was counted in a liquid scintillation counter (Beckman LS-6500). To measure PLD activity using exogenous substrate, assays were carried out with phosphatidylethanolamine (PE)/PIP(2)/[^3H]DPPC essentially according to the method of Brown et al.(9) .

Analysis of Phosphoinositides in HL60 Membranes

myo-[^3H]Inositol-labeled HL60 membranes (50 µg of protein/assay) were prepared as described above and were incubated under the same conditions of PLD assay except butanol. Reactions were terminated by the addition of chloroform/methanol/concentrated HCl (20:40:1, v/v). Lipids were extracted and separated on Silica Gel 60 TLC plates, impregnated with 1.2% potassium oxalate in a solvent system of chloroform/methanol/28% ammonia water/water (45:40:5:8, v/v) as described previously(24) . The plates were exposed to iodine vapor, and [^3H]PIP(2) was identified by comigration with PIP(2) standard. The radioactivity of spots was counted as described above. In another set of experiments, HL60 membranes (50 µg of protein) were incubated with [-P]ATP (10 µCi/ml) in the presence or absence of 0.5 mM ATP at 37 °C for 15 min. [P]PIP(2) was analyzed as above or by autoradiography.

Separation of PKC Isozymes and Small GTP-binding Proteins from Rat Brain Cytosol

Rat brains (approximately 7.0 g) were homogenized in buffer B (25 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 2 mM EGTA, 1 mM PMSF, 50 mM 2-mercaptoethanol, and 10 µg/ml leupeptin) with Polytron homogenizer (Kinematica). The homogenate was centrifuged at 100,000 times g for 60 min to obtain cytosolic fraction. The supernatant was loaded onto a Mono Q column equilibrated with buffer C (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol). Proteins were eluted with linear gradient of NaCl (0-0.7 M) in buffer C using first protein liquid chromatography (Pharmacia). The PKC activity peak eluted with 0.2-0.3 M NaCl was then applied to a Superose 12 column equilibrated with buffer C containing 150 mM NaCl and eluted with the same buffer. The PKC activity peak was pooled and then was applied to a hydroxylapatite column equilibrated with buffer D (20 mM KPO(4), pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10% glycerol, and 10 mM 2-mercaptoethanol). Proteins were eluted with linear gradient of potassium phosphate (20-300 mM) in buffer D. Individual fractions obtained from each chromatography were subjected to Western blot analysis using antibodies against PKCalpha, -betaI, -betaII, and - isozymes and small GTP-binding proteins.

Assay of Protein Kinase C Activity

The PKC activity was assayed as described previously (25) by using myelin basic protein (MBP) as a substrate(26) . The reaction mixture (50 µl) contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl(2), 2 mM CaCl(2), 10 nM PMA, 50 µg/ml phosphatidylserine, 10 µM [-P]ATP, and 50 µg/ml MBP. After incubation at 30 °C for 5 min, reactions were terminated with 50 µl of 20 mM ATP. A 50-µl aliquot was spotted on P81 phosphocellulose paper, and then the paper was washed with 75 mM phosphoric acid. The radioactivity retained on the paper was determined by Cerenkov counting. One unit of PKC was expressed as 1 pmol of [-P]ATP incorporated into MBP/5 min.

Translocation of PKCs and Small GTP-binding Proteins to Membrane

After incubation under the same conditions as the PLD assay, the reaction mixture was centrifuged at 100,000 times g for 30 min to obtain the membrane pellet. Membranes were washed once in buffer E (20 mM Tris-HCl, pH 7.4, 10 mM EGTA, 2 mM EDTA, 5 mM dithiothreitol, 1 mM PMSF, and 10 µg/ml leupeptin) and resuspended in buffer E containing 1% Triton X-100. After incubation at 4 °C for 60 min, the suspension was centrifuged at 100,000 times g for 60 min to obtain the membrane extract. Aliquots mixed with Laemmli's sample buffer (27) were subjected to electrophoresis and Western blot analysis.

Electrophoresis and Western Blot Analysis

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8 or 12% polyacrylamide gels for PKC or small GTP-binding protein, respectively, according to the method by Laemmli(27) . Proteins were electrophoretically transferred onto nitrocellulose membrane(28) . Blocking was performed in Tris-buffered saline containing 5% skimmed milk powder and 0.05% Tween 20. Western blot analysis using specific antibodies was performed as described previously(29) .


RESULTS

Requirement of Cytosolic Components for Activation of Membrane-bound PLD in HL60 Cells

Recent reconstitution studies in the cell-free system and cytosol-depleted permeabilized cells have shown that several cytosolic factors are required for activation of PLD by GTPS and/or PMA(2) . We examined the effects of cytosolic fractions separated from HL60 cells or rat brain on the membrane-bound PLD activity from HL60 cells in response to PMA and/or GTPS (Fig. 1). Weak PLD activity was detected in both cytosolic fractions, when measured using the PE/PIP(2)/[^3H]DPPC (16:1.4:1) substrate system as described by Brown et al.(9) . However, the cytosolic PLD activity was stimulated at most 2-fold with both 100 nM PMA and 10 µM GTPS (data not shown). In the absence of cytosolic fraction, little membrane-bound PLD activity was detected in response to either PMA or GTPS. However, when the cytosolic fractions from HL60 cells or rat brain were included in the reaction mixture, nearly 5-fold enhancement of PLD activity was seen in response to either 100 nM PMA or 10 µM GTPS. Furthermore, in the presence of both PMA and GTPS, the enhancement (more than 15-fold) of membrane-bound PLD activity was much greater than that observed in response to either stimulant alone.


Figure 1: Activation of PLD in HL60 membranes by cytosolic fractions. [^3H]Oleic acid-labeled HL60 membranes (50 µg of protein) and cytosolic fractions (50 µg of protein) from HL60 cells or rat brain were incubated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min in the presence of 0.3% butanol. PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of two different experiments, each carried out in duplicate.



The formation of phosphatidylalcohol such as PBut is commonly utilized to monitor PLD activity(1, 2) . Although [^3H] PBut has been reported to be metabolically stable, its degradation in various assay conditions is not clearly known. We have then analyzed the degradation of [^3H]PBut during the course of experiments. [^3H]PBut was prepared from PMA-stimulated intact HL60 cells. The level of [^3H]PBut decreased only very little (less than 10%; statistically not significant) in the reaction buffer during incubation (15 min). Additionally, membrane fraction, cytosolic fraction, and stimulators (PMA and GTPS) were almost without effect (statistically not significant) on PBut degradation in our incubation condition. Therefore, we examined PLD activity by the formation of [^3H]PBut.

Resolution of PMA-dependent PLD Activating Fractions from Rat Brain Cytosol

The PMA- or GTPS-induced PLD activation in HL60 membranes was dependent on rat brain cytosol (Fig. 2A). To separate the PMA- and GTPS-dependent cytosolic factors responsible for membrane-bound PLD activation, the brain cytosol was subjected to Mono Q anion exchange chromatography (Fig. 3A). The fractions enhancing PMA- and/or GTPS-induced PLD activity in HL60 membranes showed a broad peak. Unadsorbed fractions were ineffective in stimulating PLD activity. The active peak enhancing the PMA-induced PLD activity (fractions 35-55) was found to overlap with the peak of PKC activity (Fig. 3B). However, column fractions 32-41 also could augment the GTPS-induced PLD activity (Fig. 3A). A small GTP-binding protein RhoA was detected in these fractions by Western blot analysis (Fig. 3A, inset). The fractions 42-55 (PKC-rich fractions) were pooled and concentrated. However, this PKC-rich fraction (Mono Q-PKC) still had an ability to increase GTPS-induced PLD activity (Fig. 2B), suggesting the coexistence of small GTP-binding proteins.


Figure 2: Activation of PLD in HL60 membranes by the partially purified PKC fraction. [^3H]Oleic acid-labeled HL60 membranes (50 µg of protein) and the indicated concentrations of rat brain cytosol (A), the Mono Q-PKC fraction (B), or the Superose-PKC fraction (C) were incubated with 100 nM PMA or 10 µM GTPS at 37 °C for 15 min in the presence of 0.3% butanol. PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean of two different experiments.




Figure 3: Mono Q anion exchange chromatography of rat brain cytosol. Rat brain cytosol was subjected to Mono Q anion exchange chromatography, and each fraction was assayed for its ability to reconstitute HL60 membrane PLD activity and PKC activity as described under ``Experimental Procedures.'' A, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) and 10 µl of each fraction were incubated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min in the presence of 0.3% butanol. Western blot analysis of RhoA is shown in the inset. B, PKC activity of each fraction was measured using MBP as a substrate in the presence of 2 mM CaCl(2), 10 nM PMA, and 50 µg/ml phosphatidylserine.



In order to separate PKCs from small GTP-binding proteins, the Mono Q-PKC fraction was subjected to Superose 12 gel filtration chromatography. The PKC fraction eluted at a position of about 80 kDa was separated from the fraction containing small GTP-binding proteins (less than 80 kDa) and concentrated. This PKC fraction (Superose-PKC) contained alpha, betaI, betaII, and isozymes, but Rho family small GTP-binding proteins, RhoA, Rac1/Rac2, and Cdc42Hs were undetectable by Western blot analysis (data not shown). The Superose-PKC fraction stimulated PLD activity in response to PMA but failed to fulfill the stimulatory effect of GTPS (Fig. 2C). PLD activity was not detectable in this PKC fraction using the PE/PIP(2)/[^3H]DPPC (16:1.4:1) substrate system (data not shown) as described by Brown et al.(9) , although crude rat brain cytosol contained weak PLD activity as described above.

Activation of Membrane-bound PLD of HL60 Cells by Partially Purified PKC from Rat Brain

To examine the mechanism of PKC-mediated PLD activation, isolated HL60 membranes and the Superose-PKC fraction obtained after gel filtration were used. In the presence of the Superose-PKC fraction, PLD activation by PMA was concentration-dependent and reached the maximal level at around 100 nM PMA (Fig. 4A). An inactive phorbol ester, 4alpha-PDD failed to activate PLD (Fig. 4A). The PKC-mediated PLD activation was time-dependent and reached a plateau at 10-15 min after addition of the PKC fraction (Fig. 4B). Incubation of the HL60 membranes with the PKC fraction in the presence of 100 nM PMA caused translocation of PKCalpha, -betaI, and -betaII isozymes to membranes as inferred by Western blot analysis (Fig. 4C). The time course of PKC translocation (Fig. 4D) showed a good correlation with PMA-induced PLD activation (Fig. 4B).


Figure 4: Activation of PLD in HL60 membranes by the Superose-PKC fraction and the translocation of PKCs to membranes. A, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) and the Superose-PKC fraction (7.5 units/assay) were incubated with the indicated concentrations of PMA or 4alpha-PDD at 37 °C for 15 min in the presence of 0.3% butanol. B, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) and the Superose-PKC fraction (7.5 units/assay) were incubated with 100 nM PMA at 37 °C for the indicated times in the presence of 0.3% butanol. PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean of two different experiments. C, the HL60 membranes (50 µg of protein) and the Superose-PKC fraction (7.5 units/assay) were incubated with 100 nM PMA at 37 °C for the indicated times. Proteins in the HL60 membrane were electrophoresed and transferred to nitrocellulose membrane. Western blot analysis was performed as described under ``Experimental Procedures.'' D, quantification of relative amounts of PKCs was performed by scanning the spots on the film with a densitometer (Densitograph, ATTO).



Effect of MgATP and Phosphatidylinositol 4,5-Bisphosphate on PKC-mediated PLD Activity

The enhancement by MgATP of PLD activation in response to GTPS was demonstrated in many types of cells(15, 30, 31, 32, 33, 34) . The effect of MgATP on PKC-mediated PLD activation was examined in HL60 membranes. Although no stimulatory effect on the PKC-mediated PLD activity was observed at concentrations less than 50 µM, MgATP greatly potentiated PKC-mediated PLD activity, and the maximal effect was obtained at 0.5 mM (Fig. 5A). Recent reports (9, 35) have indicated evidence that PIP(2) functions as a cofactor for PLD activity. We examined the effect of MgATP on the level of [^3H]PIP(2) in membranes prepared from myo-[^3H]inositol-labeled HL60 cells. As shown in Fig. 5B, the level of [^3H]PIP(2) decreased in the absence of MgATP. In contrast, 0.5 mM MgATP caused an increase in the level of [^3H]PIP(2). PMA alone or both PMA and partially purified PKC fraction had no stimulatory effects on the level of [^3H]PIP(2). Additionally, the incubation of HL60 membranes with [-P]ATP in the presence of 0.5 mM MgATP led to incorporation of P into PIP(2) (data not shown). Similar observations have been observed in rat brain membranes (36) and permeabilized U937 cells(37) . These results suggest that the effect of MgATP is likely due to its ability to maintain the level of PIP(2).


Figure 5: Effect of MgATP on the PKC-mediated PLD activation in HL60 membranes. A, [^3H]oleic acid-labeled HL60 membranes were washed once in MgATP-free buffer A. The washed membranes (50 µg of protein) and the Superose-PKC fraction (7.5 units/assay) were incubated with 100 nM PMA at 37 °C for 15 min in the presence of the indicated concentrations of MgATP. PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of two different experiments, each carried out in duplicate. B, myo-[^3H]inositol-labeled HL60 membranes (50 µg of protein) were incubated with or without 0.5 mM MgATP at 37 °C for 15 min. [^3H]PIP(2) was extracted as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of three different experiments, each carried out in duplicate.



In order to further assess the involvement of PIP(2) in PMA-mediated PLD activation, we examined the effect of neomycin, which binds polyphosphoinositides with high affinity. However, neomycin is reported to inhibit PKC activity at higher concentrations (more than 2 mM)(38) . Therefore, the HL60 membrane fraction was treated with neomycin (less than 1 mM) and then excess neomycin was washed out prior to stimulation of partially purified PKC fraction and PMA. The PKC-mediated PLD activity in HL60 membranes was suppressed in parallel to increasing concentrations of neomycin (Fig. 6). 1 mM neomycin caused a complete inhibition (approximately 95%) of the PLD activity. However, the suppressed PKC-mediated PLD activity by 1 mM neomycin was restored by addition of PIP(2) in a concentration-dependent manner (Fig. 6). In these experiments, PIP(2) was mixed with phosphatidylethanolamine (PIP(2)/PE, 1:5 mol/mol), because of effective incorporation into membranes. PE alone had no effect on restoring PLD activity in neomycin-treated membranes (data not shown).


Figure 6: Effects of neomycin and PIP(2) on the PKC-mediated PLD activation in HL60 membranes. [^3H]Oleic acid-labeled HL60 membranes were incubated with the indicated concentrations of neomycin at 37 °C for 10 min and then washed once in KCl- and NaCl-free buffer A. The washed membranes were incubated with or without PIP(2)/PE (1:5, mol/mol) liposomes containing indicated concentrations of PIP(2) on ice for 30 min and then stimulated with 100 nM PMA at 37 °C for 15 min in the presence of the Superose-PKC fraction (7.5 units/assay). PLD activity was determined to measure the formation of [^3H] PBut as described under ``Experimental Procedures.'' The responses were expressed as percentages of the result obtained in the absence of neomycin and exogenous PIP(2). Data represent the mean ± S.D. of two different experiments, each carried out in duplicate.



PKC Isozymes Responsible for Activation of Membrane-bound PLD

The Superose-PKC fraction from rat brain cytosol capable of activating PLD contained alpha, betaI, betaII, and isozymes. In order to determine which isozyme was involved in PLD activation, the PKC fraction was further subjected to hydroxylapatite column chromatography; the PKC fraction after Superose 12 gel filtration was applied to a hydroxylapatite column and eluted by a linear concentration gradient of potassium phosphate. As shown in Fig. 7A, two major fractions capable of enhancing PMA-induced PLD activity were separated. Western blot analysis revealed that the first peak (peak 1) contained PKCbeta (betaI plus betaII) and PKCalpha was present in the second peak (peak 2). PKC was found in fractions 5-15 (Fig. 7B). Each PKC isozyme fraction; PKC (fractions 5-15), PKCbeta (fractions 20-35), and PKCalpha (fractions 50-70) was separately pooled and concentrated.


Figure 7: Hydroxylapatite chromatography of the Superose-PKC fraction. The Superose-PKC fraction was applied to hydroxylapatite column, and proteins were eluted with linear gradient of potassium phosphate (20-300 mM). A, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) and aliquots (15 µl) of each fraction were stimulated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min in the presence of 0.3% butanol. PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' B, proteins from each fraction were subjected to Western blot analysis with PKC isozyme-specific antibodies.



The effects of these purified PKC isozymes on HL60 membrane PLD activity were examined. In the presence of PMA (100 nM), PLD activity was enhanced by PKCalpha or -beta in a concentration-dependent manner (Fig. 8, A and B). PKCalpha was the most effective with a maximal effect obtained at about 10 units/assay. The maximal PLD activation obtained at around 10 units/assay of PKCbeta was almost half of that induced by PKCalpha. PKC had no effect on PLD activation in HL60 membranes (data not shown). The PLD activity stimulated by PKCalpha (2.5 units/assay) plus beta (2.5 units/assay) was the same as that obtained with PKCalpha alone (Fig. 8C). GTPS in the absence of PMA did not stimulate PLD activity. However, in the presence of both PMA and GTPS, the PKC-mediated PLD activity was synergistically potentiated (Fig. 8). Additionally, recombinant PKCalpha stimulated the membrane-bound PLD activity in HL60 cells in a quite similar manner as observed with the purified PKCalpha fraction (data not shown).


Figure 8: Activation of PLD in HL60 membranes by PKCalpha and beta. [^3H]Oleic acid-labeled HL60 membranes (50 µg of protein) were stimulated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min in the presence of the indicated concentrations of the PKCalpha fraction (A) and PKCbeta fraction (B). C, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) were stimulated with 100 nM PMA or both PMA and 10 µM GTPS at 37 °C for 15 min in the presence of PKCalpha (2.5 units/assay) and/or PKCbeta (2.5 units/assay). PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of two different experiments, each carried out in duplicate.



Effect of Small GTP-binding Protein RhoA on PKCalpha-mediated PLD Activation

Recently, we have demonstrated possible evidence that PKC-mediated PLD activity in HL60 membranes could be enhanced by RhoA in the presence of GTPS(20) . The PLD activity of HL60 membranes was stimulated by recombinant GTPS-bound RhoA alone in a concentration-dependent manner (Fig. 9A). RhoA at 20 nM caused a remarkable activation of the PKCalpha-mediated PLD of HL60 membranes in the presence of both PMA and GTPS (Fig. 9B). Incubation of HL60 membranes with HL60 cytosol in the presence of PMA or GTPS caused translocation of PKCalpha and RhoA to membranes, respectively, as inferred by Western blot analysis (Fig. 10). Cdc42Hs also was translocated to membranes although the extent was much less than that of RhoA (data not shown). Another Rho family member, Rac, was undetectable in either cytosol or membrane of HL60 cells as mentioned previously by Siddiqi et al.(39) .


Figure 9: Effect of RhoA on PLD activation in HL60 membranes. A, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) were stimulated with the indicated concentrations of recombinant RhoA at 37 °C for 15 min in the presence or absence of 10 µM GTPS. B, [^3H]oleic acid-labeled HL60 membranes (50 µg of protein) were stimulated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min in the presence of PKCalpha (5 units) and/or RhoA (20 nM). PLD activity was determined to measure the formation of [^3H]PBut as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of two different experiments, each carried out in duplicate.




Figure 10: Translocation of PKCalpha and RhoA to membranes in HL60 cells. The HL60 membranes (50 µg of protein) and HL60 cytosol (50 µg of protein) were stimulated with 100 nM PMA, 10 µM GTPS, or both PMA and GTPS at 37 °C for 15 min. Proteins in the HL60 membrane were electrophoresed and transferred to the nitrocellulose membrane. Western blot analysis with anti-PKCalpha antibody (A) and anti-RhoA antibody (B) was performed as described under ``Experimental Procedures.''




DISCUSSION

Several factors have been implicated in the regulation of PLD activity, such as Ca, PKC, protein-tyrosine kinase, and GTP-binding proteins(2) . However, their detailed mechanisms are not fully understood. Recently, PLD assay systems using permeabilized cells or cell-free preparations have been developed, and cytosolic factors including ARF and Rho protein are identified as regulatory factors for PLD activity. PMA, know as a PKC activator, activates PLD in many types of cells. Although the effect of PMA is assumed to be mediated through PKC, the pathway leading to PLD activation remains to be disclosed. In the present study, the mechanism of PMA-induced PLD activation was investigated in HL60 membranes.

In the cell-free system using isolated membranes from HL60 cells, the membrane-bound PLD activity was stimulated by PMA in the presence of the cytosolic fractions from HL60 cells or rat brain. The cytosolic factors required for this activity were partially purified by sequential chromatographies and identified as PKCs. Our previous report (20) has shown that the PKC-mediated PLD activation was most effectively induced in the presence of Ca. In addition, PKCalpha, -betaI, -betaII, and lesser amounts of - isozyme were observed, but PKC, -, -, -, and - isozymes were undetectable in HL60 cells by Western blot analysis (data not shown). These results suggest that conventional PKC (cPKC) isozymes may play a role in PMA-stimulated PLD activation in HL60 membranes. We have further examined PKC isozymes involved in the regulation of membrane-bound PLD. The results indicate that PKCalpha and -beta activate the membrane-bound PLD and also that PKCalpha is much more effective in its activation in HL60 cells. The study of the regulation of PLD in membranes isolated from Chinese hamster lung (CCL39) fibroblasts demonstrated that addition of purified PKCalpha and -beta from rat brain could activate PLD(8) . In our study, PKCalpha and -beta did not additively activate the PLD in HL60 membranes (Fig. 8C), suggesting that both PKC isozymes act at the same step for the PLD activation.

Previously, Tettenborn and Mueller (40) demonstrated that the PLD activation by PMA was dependent on the presence of ATP in HL60 cell lysates. Olson et al. (3) also reported that PMA-induced PLD activation was dependent on ATP in the neutrophil cell-free system. The PKC-mediated PLD activation in HL60 membranes required MgATP at millimolar concentrations (Fig. 5). Our previous study (20) has shown that the PKC-mediated PLD activation in HL60 membranes was not suppressed by Ro31-8425, a potent PKC inhibitor. Similar findings were reported by Conricode et al. (41) showing that membrane-bound PLD in CCL39 fibroblasts could be activated by PKC in a phosphorylation-independent mechanism and that the PLD activation by PKC was observed even in the absence of ATP, suggesting that PKC may activate PLD by an allosteric mechanism without ATP-dependent phosphorylation. On the other hand, it was reported more recently that the effect of ATP on PKC-mediated PLD activation is mediated by phosphorylation in human neutrophils(42) . Although this discrepancy could reflect difference in cell type, our present data cannot exclude this possibility.

Several recent studies have provided evidence that PIP(2) may act as an important cofactor for PLD activity. Brown et al. (9) developed a reconstitution system for solubilized PLD activity from HL60 cells in which the substrate PC was present in the form of mixed phospholipid micelles including PIP(2) and demonstrated the requirement of PIP(2) in the ARF-mediated PLD activation. Liscovitch et al. (35) have shown that the activity of partially purified PLD from brain membranes was stimulated considerably by PIP(2). In permeabilized U937 cells(37) , GTPS was observed to elevate the levels of polyphosphoinositides in the presence of MgATP, and either GTPS- or PMA-induced PLD activation was prevented by the antibody against phosphatidylinositol 4-kinase. Furthermore, neomycin, a high affinity ligand for PIP(2), inhibited the activity of purified PLD from brain membranes (35) and GTPS-induced PLD activation in permeabilized HL60 cells(15) , human neutrophils(17) , and U937 cells(37) . The results obtained in the present study also indicated that PIP(2) was required for PKC-mediated PLD activation in HL60 membranes. In fact, the level of PIP(2) in HL60 membranes was increased by incubation with MgATP (Fig. 5B). Therefore, MgATP is supposed to play a key role in maintaining the level of PIP(2) for PLD activity. The effect of PIP(2) might be partly explained by the fact that PIP(2) (100 µM) caused little enhancement (approximately 1.4-fold) of PKC activity (data not shown). However, at present, detailed information for the site of action of PIP(2) and involvement of protein phosphorylation is not available and should be obtained by additional experiments which are currently in progress in our laboratory.

The PKC-mediated PLD activation in HL60 membranes was potentiated by the addition of GTPS (Fig. 8). This finding led us to assume that translocated PKCalpha or -beta interacts with membrane-associated GTP-binding proteins, resulting in a synergistic activation of PLD. Several lines of evidence are present to indicate that activation of PLD is mediated by small GTP-binding proteins which interact directly with the solubilized PLD(9, 39, 43, 44, 45) . In addition, it was demonstrated that PIP(2) synthesis is regulated by Rho family small GTP-binding proteins(46, 47) . Our previous study (20) has shown that RhoGDI, Rho GDP dissociation inhibitor which extracts Rho proteins from membranes, prevented the synergistic effect by GTPS in PKC-mediated PLD activation in HL60 membranes, and also that this suppressed PLD activation was restored by the addition of recombinant RhoA. These results suggest the interaction between PKC and membrane-associated RhoA. Western blot analysis with the antibody to RhoA showed the presence of RhoA in the HL60 membrane fraction (data not shown). Furthermore, the present study showed that PKCalpha-mediated PLD activity was potentiated by the addition of recombinant RhoA in the presence of GTPS (Fig. 9). It was also shown that cytosolic PKCalpha and RhoA were translocated to membranes in a PMA- and GTPS-dependent manner, respectively. Most recently, Siddiqi et al. (39) indicated that not only RhoA but also Rac and Cdc42Hs induced PLD activation in HL60 membranes. Among the Rho family GTP-binding proteins, however, only RhoA was translocated to HL60 membranes in the presence of GTPS(39) . These findings suggest the involvement of RhoA in the regulation of membrane-bound PLD in HL60 cells.

Despite many studies, the exact mechanism for the implication of PKC in PLD activation has not yet been defined. It may be possible that PKC interacts directly with PLD in membranes or that PKC interacts with other membrane-associated proteins that in turn activates PLD. In the present study, the PKCalpha fraction free from small GTP-binding proteins was observed to activate the membrane-bound PLD (Fig. 8). However, GTPS synergistically enhanced PKC-mediated PLD activation. This synergistic activation by GTPS was prevented by pretreatment of HL60 membranes with RhoGDI, as shown in the previous study(20) . Recent reports demonstrate that ARF-mediated PLD activation is modulated by other as yet unidentified cytosolic proteins(48, 49, 50) . RhoA may act as a PKC-modulating factor in PKC-mediated PLD activation. We have recently obtained a preliminary finding that in the membranes pretreated with GDPbetaS, the PKCalpha-mediated PLD activation by PMA was almost completely abolished. Thus, the PKC-mediated PLD activation mechanism involving RhoA appears to be more complex than expected. The reconstitution system using purified membrane-bound PLD will add further insight into the mode of the synergistic activation of membrane-bound PLD in HL60 cells.


FOOTNOTES

*
This study was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan. 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. Tel.: 81-58-267-2228; Fax: 81-58-265-9002.

(^1)
The abbreviations used are: PLD, phospholipase D; PBut, phosphatidylbutanol; PKC, protein kinase C; PMA, 4beta-phorbol 12-myristate 13-acetate; 4alpha-PDD, 4alpha-phorbol 12,13-didecanoate; GTPS, guanosine 5`-O-(3-thiotriphosphate); GDPbetaS, guanosine-5`-O(2-thiophosphate); PC, phosphatidylcholine; PE, phosphatidylethanolamine; PA, phosphatidic acid; PIP(2), phosphatidylinositol 4,5-bisphosphate; MBP, myelin basic protein; PMSF, phenylmethylsulfonyl fluoride; ARF, ADP-ribosylation factor; GDI, GDP dissociation inhibitor; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Y. Takai (Osaka University, Japan) for the kind gift of recombinant RhoA and GST-RhoGDI.


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