©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Phospholipase D by Protein Kinase C in Human Neutrophils
CONVENTIONAL ISOFORMS OF PROTEIN KINASE C PHOSPHORYLATE A PHOSPHOLIPASE D-RELATED COMPONENT IN THE PLASMA MEMBRANE (*)

(Received for publication, January 12, 1995; and in revised form, June 16, 1995)

Isabel Lopez (1) David J. Burns (2) J. David Lambeth (1)(§)

From the  (1)Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322 and (2)Sphinx Pharmaceutical, Durham, North Carolina 27707

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In a variety of intact cells, phorbol esters are known to activate phospholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATPS (adenosine 5`-O-(thiotriphosphate)) was 2-3-fold more effective than ATP, while ADP and AppNHp (adenyl-5`-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol depleted of protein kinase C by chromatography on threonine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane: plasma membrane was phosphorylated using ATPS/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta(1) > alpha > ; the beta(2), , , , and isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.


INTRODUCTION

Phospholipase D (PLD) (^1)catalyzes the hydrolysis of phospholipids to generate phosphatidic acid plus the headgroup. In eukaryotic cells the enzyme is relatively specific for phosphatidylcholine. Phosphatidic acid may act directly as a signal molecule (1, 2) or can be metabolized to form diacylglycerol (DG) by phosphatidic acid phosphohydrolase. The latter can function as an activator of protein kinases C (PKC) and possibly other diacylglycerol-dependent enzymes. In the presence of primary alcohols, PLD also catalyzes a unique transphosphatidylation reaction to produce the phosphatidylalcohol (e.g. phosphatidylethanol), which can be used as a convenient measure of PLD activity(3) . PLD can be activated by a variety of agonists, including those that act via some G protein-coupled receptors and via receptor tyrosine kinases (2, 4, 5) (reviewed in (2) ). In a wide variety of cells, phorbol esters are also potent activators of PLD. In human peripheral blood neutrophils, both fMet-Leu-Phe, which acts via a G protein-coupled receptor, and phorbol esters, which presumably act through PKC, activate PLD(6, 7, 8) .

Although receptor-coupled PLD enzyme(s) have not been purified, cell-free systems have advanced the understanding of the enzymology and regulation of PLD. Exton and colleagues (4) observed GTPS-activated PLD in liver plasma membranes. Cell-free reconstitution of PLD activity in neutrophil fractions requires the participation of protein components not only in the plasma membrane, but also in the cytosol(7, 9) . Activation was achieved using either GTPS or phorbol esters, and required micromolar calcium(7) . In the neutrophil system, one of the plasma membrane components is a Rho family small molecular weight GTP-binding protein(10) . RhoA has been described in liver plasma membranes as the PLD-activating GTP-binding component(11) . Another small GTP-binding protein, ARF, was purified from brain cytosol as a factor which activates the PLD in HL-60 membranes(12, 13) . Under our conditions, ARF is not essential for PLD activation, but synergizes with an essential 50-kDa cytosolic factor(14) , the identity of which is not yet clear.

PKC plays an important role in signal transduction, regulating a variety of cellular functions via phosphorylation of target proteins. PKC is comprised of a family of related enzymes that are differentially expressed in a variety of tissues and cell types(15, 16, 17) . To date, 10 PKC isoenzymes have been identified and can be categorized into three groups (conventional, novel, and atypical) based on functional properties as well as sequence. The conventional PKCs (cPKCalpha, beta(I), beta, and ) require calcium and phospholipid, and are activated by DG or phorbol ester. The novel PKCs (, , , and ) are also activated by diacylglycerol or phorbol esters and require phospholipid, but do not require calcium. On the other hand, the atypical PKCs ( and ) are not activated by DG or phorbol esters but are dependent on phosphatidylserine.

Activation of PLD by phorbol esters is assumed to occur via one or more PKCs, but other effects of phorbol esters not involving PKC have been suggested. In addition, in liver plasma membranes, PLD activation by phorbol esters, while requiring PKC, is independent of ATP, suggesting a phosphorylation-independent mechanism (18) (e.g. direct complexation of PKC with PLD). In the cell-free system from neutrophil, phorbol ester-dependent activation of PLD requires ATP and is inhibited by staurosporine(7) , suggesting a classical phosphorylation-dependent mechanism. In the present studies, we utilized biochemical approaches to investigate the function of PKC in the activation of neutrophil PLD. We find that activation requires conventional isoforms of PKC (alpha, beta(1), or ) and that the target of phosphorylation is in the plasma membrane.


EXPERIMENTAL PROCEDURES

Materials

Hespan (6.2% hetastarch in 0.9% NaCl) was obtained from Du Pont. Lymphocyte separation medium (6.2% Ficoll, 9.4% sodium diatrizoate) was purchased from Organon Teknika (Durham, NC). Purified rat brain PKC (mixture of isoforms) was obtained from Calbiochem. Phosphatidylethanol standard was from Avanti Polar Lipids (Alabaster, AL). ATP, ATPS, AppNHp, and ADP were purchased from Boehringer Mannheim. GTPS, diisopropyl fluorophosphate, 4alpha-phorbol 12-myristate 13-acetate, histone type IIIS, L-threonine, sodium orthovanadate, Tris-HCl, HEPES, EDTA, staurosporine, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and DEAE-cellulose anion exchange resin were obtained from Sigma. Silica Gel 60 TLC plates (0.25 mm thick) were acquired from EM Science (Gibbstown, NJ). 1-O-[^3H]Octadecyl-sn-glycero-3-phosphocholine (80-180 Ci/mmol) and [-P]ATP (>5000 Ci/mmol) were purchased from Amersham Corp. Okadaic acid was obtained from Life Technologies, Inc. Phorbol 12,13-dibutyrate was from LC Services Corp (Woburn, MA). Sephadex G25 gel filtration (medium) media and EAH-Sepharose 4B were acquired from Pharmacia Biotech Inc. Centricon 10 microconcentrators were purchased from Amicon (Beverly, MA). All other reagents and solvents used were of highest quality available commercially.

Preparation of Plasma Membranes and Cytosolic Fractions from Human Neutrophils

After obtaining informed consent, human neutrophils were isolated from venous blood of adult donors(19) . Isolated cells were resuspended in HEPES/saline buffer (25 mM HEPES, 125 mM NaCl, 0.7 mM MgCl(2), 0.5 mM EGTA, 10 mM glucose, and 1 mg/ml fatty acid-free bovine serum albumin, pH 7.4) at a concentration of 2 10^7 cells/ml and used immediately. Cells were labeled with [^3H]1-O-alkyl lysophosphatidylcholine (1.5 µCi/ml)(20) . After labeling, the cells were treated with 4 mM diisopropyl fluorophosphate, pelleted at 600 g for 7 min at 4 °C, and resuspended in incubation buffer (25 mM HEPES, 100 mM KCl, 3 mM NaCl, 5 mM MgCl(2), pH 7.4, containing 2 mM each leupeptin, aprotinin, and pepstatin and 0.5 mM phenylmethylsulfonylfluoride). The cells were disrupted by nitrogen cavitation (pressurized at 450 p.s.i. for 20 min at 4 °C). Plasma membranes and cytosolic fractions were isolated as described previously(7) . Unless specified the cytosol used in these studies was chromatographed after isolation on a Sephadex G25 gel filtration column (36 1.4 cm, 1 ml/fraction) at 4 °C to remove endogenous free nucleotides. Cytosol treated in this manner is referred to as ``nucleotide-depleted cytosol.''

Phospholipase D Assay and Lipid Extraction

PLD incubations and assays were carried out according to Olson et al.(7) . Test tubes containing incubation components were kept on ice until all reagents were added. Tubes were then placed in a 37 °C shaking water bath for 20 min, and the reaction was terminated by transferring contents to glass tubes containing 1.5 ml of CHCl(3)/MeOH (1:2, v/v). Lipids were extracted according to Bligh and Dyer (21) except that 2% acetic acid was used instead of water. The chloroform phase was dried under vacuum, and the resulting lipid pellet was solubilized in CHCl(3)/MeOH (95:5, v/v). Samples were then spotted onto Silica 60 TLC plates and developed as described(7) . Phosphatidylethanol (R = 0.7) was used as standard and visualized by iodine vapor. Radioactivity in the labeled lipids was quantified using a Bioscan System 200 imaging scanner. PLD activity is expressed as percentage of counts in the phosphatidylethanol spot compared to total counts in all spots in the same lane.

Preparation of Threonine-Sepharose

Threonine-Sepharose was prepared according to Go et al.(22) . Briefly, EAH-Sepharose 4B was washed several times with deionized H(2)O and resuspended in 15 ml of H(2)O. L-Threonine (3 g in 10 ml of H(2)O) and 1-ethyl-3-(3-dimethylamine)carbodiimide hydrochloride (3 g in 5 ml of H(2)O) were then added to the gel suspension and mixed end over end on a rotating wheel overnight at room temperature. The pH of the reaction was maintained at 5-6 during the reaction. The gel was then washed alternately several times with 600 ml each of pH 8.0 (0.1 M NaHCO(3), 0.5 M NaCl) and pH 4.0 (0.1 M NaC(2)H(3)O(2), 0.5 M NaCl) buffers to remove excess ligand, urea derivative, and unreacted carbodiimide. Finally, the gel was washed with 500 ml of distilled H(2)O followed by a wash with 20 mM Tris-HCl buffer (pH 7.4; containing 5 mM MgCl(2), 20 mM KCl, and 5 mM mercaptoethanol).

Depletion of PKC from Cytosol

It has been shown previously that PKC binds to threonine-Sepharose(22) , and this methodology was used with slight modifications to deplete cytosol of PKC. Cytosol (30-60 mg in 20 ml) was added to a 10-ml slurry of DEAE-cellulose in a conical 50-ml centrifuge tube and placed on a revolving wheel to mix for 15 min. The DEAE-cellulose had been previously equilibrated with 20 mM Tris-HCl at pH 7.4, containing 100 mM KCl, 5 mM MgCl(2), 3 mM NaCl, and 5 mM mercaptoethanol. The cytosol DEAE-cellulose slurry was then centrifuged and the cytosol layer transferred to another tube. The cytosol was further diluted with 20 mM Tris-HCl at pH 7.4 (containing 5 mM MgCl(2)) to lower the KCl concentration to 20 mM. Diluted cytosol was then applied to the threonine-Sepharose column (1.5 18 cm, 4 ml/fraction), which had been previously equilibrated with 20 mM Tris-HCl at pH 7.4, containing 20 mM KCl, 5 mM MgCl(2), and 5 mM mercaptoethanol (Buffer A). The column was washed with 60 ml of buffer A, and PKC was eluted with Buffer A containing 1 M NaCl. Fractions were assayed for PLD activity (above) and for PKC activity by measurement of P incorporation from [-P]ATP into histone type-IIIS in the presence of 40 µM phosphatidylserine and 500 µM CaCl(2)(23) . Adjacent fractions were pooled, concentrated using a Centricon 10 microconcentrator, and frozen at -80 °C prior to assay. PKC depleted cytosolic fractions were further pooled and concentrated at the time of the assay. Protein was determined by the Bradford method (24) using bovine serum albumin as the protein standard.

Immunoblotting

Samples were subjected to 12% SDS-polyacrylamide gel electrophoresis and were electrophoretically transferred to nitrocellulose at 4 °C. Nonspecific binding sites on nitrocellulose were blocked by incubation with 10% nonfat dry milk in 1 Tris-buffered saline-Tween (TBS-T; containing 20 mM Tris base, 137 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 h at room temperature. The nitrocellulose sheets were then incubated for 2 h at room temperature with rabbit antisera (1:1000 dilution) to peptides specific to PKC isoforms, kindly provided by Dr. Yusuf Hannun (Duke University, Durham, NC). These antibodies have been previously described and characterized(25) . After three washes with 1% nonfat milk in TBS-T at 10-min intervals, alkaline phosphatase-conjugated second antibody (goat anti-rabbit at 1:2000 dilution) was added and incubated for 2 h at room temperature. The nitrocellulose sheet was then subjected to three 10-min washes with 1% nonfat dry milk, followed by three 1-min washes and one 5-min wash with TBS. Color was developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate(26) .

Pretreatment and Reisolation of Plasma Membranes

Isolated plasma membranes (250-300 µg) were incubated in the presence of nucleotide-depleted cytosol (500-600 µg) or purified PKC, phorbol 12,13-dibutyrate (PDBu; 1 µM), and ATPS (1 mM) in buffer containing 5 µM CaCl(2) for 5 min at 30 °C. After the preincubation, the reaction mixture was layered on a discontinuous sucrose gradient (400 µl each of 20% and 60% sucrose) and centrifuged at 260,000 g for 20 min at 4 °C in a Beckman TL-100 ultracentrifuge. After discarding the upper layer, the plasma membrane was recovered from the interface between the sucrose layers, homogenized, and utilized in PLD assays.


RESULTS

Adenine Nucleotide Requirement for Phorbol Ester Stimulation of PLD

In liver plasma membranes, phorbol ester activation of PLD is independent of ATP(18) . Using neutrophil plasma membranes and nucleotide-depleted cytosol, we quantified the effect of PMA on PLD activity, measured by the formation of [^3H]phosphatidylethanol (PEth). As shown in Fig. 1, while 1 mM ATP alone did not activate, PMA-dependent activation of PLD was dependent upon the presence of ATP, confirming our earlier report(7) . However, as was seen in the earlier study, activation by PMA/ATP was roughly only half of that seen using GTPS. A significantly greater response was seen when 1 mM ATPS was used in place of ATP during phorbol ester activation. In several experiments, the response to PMA/ATPS was typically as large as or slightly larger than the response to GTPS. In addition, the responses to PMA/ATPS and GTPS were non-additive (data not shown), suggesting that they are activating the same response. Since ATPS can serve as a kinase substrate for thiophosphoryl transfer, AppNHp was also tested. This analog, which cannot be utilized by protein kinases, was ineffective, as was ADP.


Figure 1: Adenine nucleotide specificity for PMA stimulation of phospholipase D. Isolated plasma membranes (25 µg) and nucleotide-depleted cytosol (50 µg) were incubated in buffer containing 1 µM CaCl(2) for 20 min at 37 °C with either 10 µM GTPS or with 100 nM PMA in the presence or absence of 1 mM each of the following adenine nucleotides: ADP, ATP, ATPS, or AppNHp as detailed under ``Experimental Procedures.'' [^3H]Phosphatidylethanol was quantified as described under ``Experimental Procedures'' and is expressed as the percentage of total counts in lipids. Basal PEth in the absence of activators or nucleotides was 1 ± 0.2%, and this value was subtracted from the values shown. Each value is the mean ± S.E. of three to five experiments, each using a separate preparation of plasma membrane and cytosol performed in duplicate.



The dose dependence for adenosine nucleotides in PMA-activated PLD is shown in Fig. 2. PMA in the absence of nucleotides failed to activate. ATP showed a broad concentration optimum (10 µM to 1 mM), while that for ATPS was 1 mM. The maximal response with ATP, however, was only about one third of the maximal response seen with ATPS. The latter response in this experiment was similar to that seen with GTPS (filledtriangle, lowerpanel). Staurosporine (1 µM), a protein kinase inhibitor, almost completely inhibited PMA stimulated PLD activity at all concentration ranges of both ATP and ATPS. Thus, these data are consistent with both ATP and ATPS serving as phosphate donors, activating PLD in a PKC-dependent phosphorylation reaction. In addition, when [S]ATPS was used, a large number of proteins in the plasma membrane were phosphorylated in a PMA-dependent manner, indicating facile transfer of the thiophosphoryl group via PKC (data not shown).


Figure 2: Concentration dependence of ATP or ATPS for PMA stimulation of PLD and inhibition by staurosporine. Isolated plasma membranes (25 µg) and nucleotide-depleted cytosol (50 µg) were incubated in buffer containing 1 µM CaCl(2) with 10 either µM GTPS (filledtriangle, lowerpanel) or with 100 nM PMA plus the indicated concentration of either ATP (upperpanel, filledcircles) or ATPS (lowerpanel, filledcircles) as detailed under ``Experimental Procedures.'' ATP or ATPS alone were also varied in the upper and lowerpanels, respectively, and PLD activity measured (opencircles). Staurosporine (1 µM) was included along with ATP or ATPS in the upper and lowerpanels (opentriangles), respectively. Reactions were incubated for 20 min at 37 °C and terminated, and PEth was quantified as described under ``Experimental Procedures.'' Basal PEth formed in the absence of activators and inhibitors was 0.5 ± 0.2% (diamond, lowerpanel). Each value represents the mean ± range of two experiments each using separate preparations of cell fractions, with each point in each experiment the average of duplicate determinations.



The likely explanation for the enhanced effectiveness of ATPS compared with ATP is that the thiophosphoryl group, once transferred to the protein, is resistant to the action of phosphatases, thereby increasing the stability of the phosphorylated form of the protein. Dubyak and colleagues (27) showed that in a cell-free system from U937 cells, inhibitors of protein phosphatases such as vanadate augmented the activation of PLD. As shown in Fig. 3, under our conditions, neither vanadate nor okadaic acid augmented PMA-stimulated activity when either ATP or ATPS were used. Dose dependence curves for these compounds were also carried out (not shown), and enhancement of activity with ATP was not seen at any concentration used. Thus, phosphatase inhibitors in this system did not appear to be useful for preserving the activated state.


Figure 3: Effects of okadaic acid and vanadate on PMA-stimulated phospholipase D activity. Isolated plasma membranes (25 µg) and nucleotide-depleted cytosol (50 µg) were incubated in buffer containing 1 µM CaCl(2) as indicated either with 10 µM GTPS or with/without 100 nM PMA in the presence of 1 mM ATP or 1 mM ATPS. Okadaic acid (10 µM) or vanadate (100 µM) were also included as indicated, and the reaction mixture was incubated for 20 min at 37 °C. Reactions were terminated and PEth quantified as described under ``Experimental Procedures.'' PEth was 1.6 ± 0.5% in the absence of any added activators or inhibitors, and this value was subtracted from those shown. Each value represents the mean ± range of two experiments, each carried out with a separate preparation of subcellular fractions and done in duplicate.



The increase in [^3H]PEth seen with PMA/ATPS was apparently due exclusively to stimulated formation, and there was no effect on its subsequent metabolism, which is generally assumed to be minimal. To verify this assumption, we included 0, 40, and 400 nM unlabeled PEth in an incubation. This represents about 10- and 100-fold more PEth than would be formed during the incubation. If PEth were unstable in the incubation mixture, then cold PEth should compete for metabolism, thereby enhancing the recovery of the [^3H]PEth formed during the incubation. In fact, unlabeled PEth added to the incubation had no effect (data not shown).

Reconstitution of PLD Activity by PKC

To demonstrate conclusively that PKC was responsible for PMA-dependent activation of PLD, PKC was depleted from neutrophil cytosol using DEAE-cellulose and threonine-Sepharose chromatography as detailed under ``Experimental Procedures.'' The latter column chromatography is shown in Fig. 4A. The fractions obtained were tested for both PKC and PLD activities. All of the PKC activity, as determined by [P]phosphate incorporation into histone (type III-S), appeared as a single peak between fractions 27 and 30 (Fig. 4A, inset). Because of the relative insensitivity of the PLD assay, fractions were pooled and concentrated, as indicated in Fig. 4A. Upon recombination of these fractions with plasma membranes, approximately 70% of the integrated GTPS-stimulated PLD activity (the summation of activity times volume) eluted as a broad peak in fractions 1-22 (openbars, Fig. 4A, referred to as PKC-depleted cytosol), with additional activity co-eluting in fraction 27-30 along with PKC. Aliquots from pooled fractions were also stimulated with PMA/ATPS in the presence of plasma membrane. While PMA-supported PLD activity was reconstituted using pooled fractions 27-30, no detectable PMA-supported PLD activity was seen using earlier fractions. These fractions also supported GTPS-dependent PLD activity. Thus, removal of PKC eliminates phorbol ester-dependent activation, but guanine nucleotide-dependent activation is retained.


Figure 4: Separation of PKC from neutrophil cytosol by column chromatography. PanelA, neutrophil cytosol (60 mg protein) was chromatographed at 4 °C using DEAE followed by threonine-Sepharose column, as detailed under ``Experimental Procedures.'' At the arrow (inset), buffer containing 1 M NaCl was added. Fractions (4 ml) were collected, and an aliquot of each was analyzed for PKC activity (inset), using histone phosphorylation as described under ``Experimental Procedures.'' Fractions were pooled as indicated, concentrated, and assayed for PLD-supporting activity as detailed under ``Experimental Procedures,'' using either 10 µM GTPS or 100 nM PMA plus 1 mM ATPS as activators in the presence of 1 µM CaCl(2). PanelB, samples were analyzed for the indicated isoforms of protein kinase C, using immunoblotting with isoform-specific antisera, as described under ``Experimental Procedures.'' Lanes are as follows: lane1, neutrophil cytosol (60 µg); lane2, pooled fractions 1-22 (60 µg); lane3, pooled fractions 27-30 (60 µg); lane4, 1 µg each of purified recombinant protein kinase Calpha, beta(1), beta(2), , and , respectively (top to bottom); lane5, 1 µg each of purified recombinant protein kinase C beta(1), , alpha, , , respectively (top to bottom, used to demonstrate antibody specificity).



To verify that all isoforms of PKC had been removed from the ``PKC-depleted cytosol,'' immunoblotting was carried out on cytosol, on PKC-depleted cytosol (fractions 1-22) and on the PKC-containing pooled material (fractions 27-30). No PKC isoforms were detected in the PKC-depleted pool. However, alpha, beta(1), beta(2), and isoforms were detected in both the cytosol and in pooled fractions 27-30 (Fig. 4B). The isoform was detected at a low level in neutrophil cytosol (Fig. 4B), but was not seen in fractions 27-30, indicating that this isoform remained associated with the threonine-Sepharose column. PKC , , and were not detected, consistent with other reports.

Fractions 1-22 were pooled, concentrated, and used to determine if PLD activity could be reconstituted with purified rat brain PKC. As shown in Table 1, PMA/ATPS-stimulated PLD activity was seen when plasma membranes were incubated with native cytosol, but no activity was seen when PKC-depleted cytosol was used. However, when plasma membranes were incubated with PKC-depleted cytosol plus purified rat brain PKC (a mixture of isoforms), PMA/ATPS-stimulated PLD activity was recovered to a level slightly higher than that seen with native cytosol.



Localization of the PKC Target

The target of phosphorylation was localized to the plasma membrane using a 5-min prephosphorylation protocol. Plasma membranes were treated for 5 min with PDBu plus ATPS in the presence of either nucleotide-depleted cytosol (as a source of PKC) or purified rat brain PKC itself. (^2)PDBu rather than PMA was used, since the former is water-soluble and can be more easily removed from membrane systems than PMA. Following membrane reisolation by discontinuous sucrose gradient centrifugation to remove activating factors, membranes were recombined with nucleotide-depleted cytosol and assayed for PLD activity. As shown in Table 2(middle data column), when the reisolated membranes had been pretreated to (thio)phosphorylate a protein target in the membrane, PLD activity was seen upon addition of nucleotide-depleted cytosol in the absence of any additional agonist. No activity was seen in any case when cytosol was omitted (left data column).



Membranes preincubated with PDBu/ATPS alone did not show significant activity upon recombination with cytosol, indicating that carryover of the activating factors into the incubation was not a problem. Using radiolabeled [S]ATPS in the incubation/reisolation protocol, 0.6% was carried over into the plasma membrane fraction, and using [^3H]PDBu, 3% was recovered with plasma membranes. Thus, the final concentration of ATPS carried over into the second incubation was 6 µM, well below the concentration needed to support phorbol ester-dependent activation (see Fig. 2). Although a potentially activating concentration of PDBu (30 nM) may have been carried through, this was not a problem since the ATPS was effectively removed. Also, preincubation with cytosol alone did not permit activator-independent activation. As a positive control to demonstrate that the PLD activity was still present after reisolation of plasma membranes, these preparations were stimulated with PMA/ATPS in the presence of added cytosol (right-hand data column, Table 2).

Calcium Dependence for PKC Activation of PLD

To determine if the activation of PLD by PKC was calcium-dependent, plasma membranes were preincubated with nucleotide-depleted cytosol in the presence or absence of PDBu plus ATPS and in the presence or absence of 5 µM calcium. The plasma membrane was then reisolated as above, to remove cytosol and activators, and was then recombined with nucleotide-depleted cytosol. Activator-independent PLD activity was seen only when plasma membranes were thiophosphorylated in the presence of both calcium and activators (see Table 3, left data column). As a control, in the right-hand column, both activators and cytosol were added, demonstrating that all groups of plasma membranes remained active (maximal activity = 5-6% PEth). Thus, the phorbol ester/PKC-dependent activation step was calcium-dependent, implicating a conventional form of PKC.



Selective Activation of PLD by PKC Isoforms

To evaluate the PKC isoforms involved in PLD activation, plasma membranes were thiophosphorylated with the same activity (determined by histone IIIS phosphorylation) of either rat brain PKC or purified recombinant human PKC isoforms. The membranes were then reisolated and recombined with nucleotide-depleted cytosol in the absence of additional activators, and PLD activity was quantified. Results are summarized in Table 4. The conventional isoforms (alpha, beta(1), and ) showed high activity, while the novel (, and ) and atypical form () showed little or no activity. Interestingly, the beta(2) isoform, a splice variant of beta(1), showed no activity.



Concentration dependence curves using highly purified preparations of the major activating isoforms were also carried out and are summarized in Fig. 5. All three of the conventional isoforms were active, but there was a demonstrable rank order of specificity for the three, with PKCbeta(1) > PKCalpha > PKC, which showed respective EC values of 0.6, 5, and 20 ng/ml, respectively. The maximal activation of PLD by all effective PKC isoforms was similar. Several concentrations in this same range of non-conventional and atypical isoforms of PKC were also tested. None of these produced significant activation. A complete concentration dependence curve was also carried out for PKC (Fig. 5, lowerpanel), which showed no activity at any concentration tested.


Figure 5: Concentration dependence for activation of phospholipase D by isoforms of protein kinase C. The concentration of PKC was varied as indicated in the preincubation (5 min at 30 °C) with plasma membrane in the presence of 1 µM phorbol 12,13-dibutyrate plus 1 mM ATPS and 5 µM CaCl(2). Plasma membranes were then reisolated on a discontinuous sucrose gradient, recombined, and incubated with nucleotide-depleted cytosol as detailed under ``Experimental Procedures.'' No further addition of activators was made to the incubation. Each experiment was repeated two to three times with duplicate incubations each time for each isoform of PKC; points shown represent the average of all determinations. The isoform for each titration is indicated in each panel.




DISCUSSION

The present study has investigated the role of PKC in the activation of neutrophil PLD. Activation of PLD by phorbol esters has been widely documented in a variety of tissues (for review, see (2) ), and it is assumed that PKC participates directly or indirectly in this process. In some cells, down-regulation of PKC elicited by chronic exposure to phorbol esters prevents activation of PLD by phorbol esters as well as by other agonists(28, 29, 30) . Nevertheless, PKC-independent mechanisms of phorbol ester activation have been suggested in some cases(31, 32, 33) . In one of the few studies carried out in a cell-free system, Conricode et al.(18) showed that phorbol ester activation in cell membranes from a lung fibroblast cell line, while dependent on PKC, was independent of ATP and protein phosphorylation. Inhibitors of PKC such as staurosporine and H-7, which act competitively at the ATP-binding site, are inhibitory toward PMA activation in some systems(7, 34, 35) , while failing to inhibit in others(29, 34) . Phosphorylation-independent activation suggests a direct complex formation between PKC and a PLD-related protein. In this regard, a protein ``receptor'' for PKC has been described recently (37) , and such a binding target might be related to PLD activation.

Studies in neutrophils, however, support a classical picture of PKC action involving phosphorylation of a target protein. Previous studies in our laboratory (7) and others (38) have demonstrated that PLD activation by phorbol esters required ATP. Nevertheless, activation by phorbol esters in the presence of ATP was consistently less effective than that by GTPS. In the present studies, which utilize a cell-free system, we confirm that phorbol ester activation requires ATP or an ATP analog. In addition, we find that when ATPS was used in place of ATP, phorbol esters activated to an extent as great or greater than GTPS. Consistent with a phosphorylation mechanism, staurosporine inhibited PMA activation when either ATP or ATPS were used but did not inhibit activation by GTPS(7) . (^3)This rules out the possibility that ATPS might be acting via a nucleoside diphosphate kinase to generate GTPS. Thus, the most likely mechanism for the enhanced effectiveness of ATPS over ATP is that the thiophosphoryl group, once transferred to the protein target, is more stable to the effects of endogenous phosphatases than is the phosphate group, as has been seen in other studies. Some protein phosphatase inhibitors were also tested for possible stimulatory effects when ATP was used. Vanadate is a tyrosine phosphatase inhibitor, while okadaic acid inhibits protein phosphatase 1 and 2A. Both inhibitors have been shown previously to augment phorbol ester-stimulated PLD activity in U937 cells and in intact and permeabilized neutrophils(39, 40, 41) . However, neither of these inhibitors enhanced the PMA-stimulated PLD activity in our cell-free system. This may indicate that other phosphatases play a role in this setting and/or that the effects seen by other workers may be due to phosphorylations higher up in the activation pathway.

Further support for a classical mechanism for phorbol ester/PKC activation came from studies in which PKC was depleted from the cytosol with loss of PMA-dependent activity (Fig. 4A and Table 1). Interestingly, activation by GTPS was retained, indicating that the activation by the GTP-binding protein does not require the direct participation of PKC. Addition of rat brain PKC to the depleted system reconstituted activity (Table 1). Activity retained its absolute dependence on cytosol, indicating that the cytosolic factor was not PKC itself. We have recently found that the cytosolic factor is an approximately 50-kDa protein, and that this factor synergizes with ADP-ribosylation factor in the activation of PLD(14) .

Major isoforms of PKC that are present in human neutrophils include conventional (alpha, beta(1), and beta(2)), novel ( and ), and atypical () forms (42, 43, 44) , (^4)and are indicated in Table 4. Our studies confirm the presence of these isoforms in neutrophil cytosol. In addition, we show that these isoforms are completely removed from cytosol by threonine-Sepharose chromatography, thus enabling us to reconstitute the system with individual PKC isoforms. The present studies implicate conventional (calcium-dependent) but not other isoforms of PKC in the activation of neutrophil PLD. Our preincubation/reisolation protocol allowed us to separately evaluate the calcium requirement of the PLD-activating PKC isoforms present in neutrophil cytosol and the calcium dependence of the PLD itself. This was important, since both PMA- and GTPS-dependent activation require a submicromolar concentration of calcium, which is presumably due to a calcium requirement for the phospholipase itself(7) . Using this two-stage protocol, we found that the activating PKC present in cytosol was calcium-dependent, implicating a conventional isoform. This was confirmed using purified recombinant PKC isoforms (Table 4). Only the conventional isoforms resulted in significant activation. When dose responses were carried out, the PLD activation showed the following rank order of specificity for PKC: PKCbeta(1) > PKCalpha > PKC. Interestingly, the beta(2) isoform, which differs from PKCbeta(1) by 50 residues at the C terminus, was completely inactive. Our studies are in agreement with studies in rat fibroblasts in which PKCbeta(1) overexpression was accompanied by an increase in PMA-activated PLD(45, 46) . Overexpression of PKCalpha in Swiss/3T3 fibroblasts also resulted in enhanced PLD activity, but this was attributed to increased induction of the lipase itself(47) . In membranes from a lung fibroblast cell line, Exton and colleagues (48) also found that only conventional forms of PKC activated, but in this case, the effect of alpha was greater than beta, and showed no activity. Notably, in contrast to our cell-free system, phorbol ester/PKC activation did not require protein phosphorylation and occurred in the absence of cytosol(18) . Insel and colleagues (49) used antisense technology to reduce levels of either PKC alpha or beta in Madin-Darby canine kidney cells. Elimination of the alpha but not the beta form resulted in a 70% loss in PLD activity. In contrast, several in vivo studies (50, 51, 52) that were based on correlative data or on selective down-regulation of PKC isoforms have implicated non-conventional forms of PKC, in particular PKC. Although the neutrophil is not known to contain this isoform, we investigated its function, both at a single concentration (Table 4) and in a dose dependence curve (Fig. 5). No activity was seen at any concentration used, indicating that the neutrophil PLD cannot be functionally linked to this isoform. Finally, PLD activity has been reported in one case to be inhibited through the action of PKC(53) . Notably, in our studies, the PKCalpha (and, to a lesser extent, ) showed inhibitory phases at higher levels of the kinase. This may indicate that there are not only activating but also inhibitory phosphorylations by some isoforms of PKC. Differences seen in PKC isoform selectivity may reflect different isoforms of PLD that are likely to be present in different tissues(2) .

Because reconstitution of PLD activity in neutrophil requires both plasma membrane and cytosol, it was important to define which fraction contained the target for PKC phosphorylation. Using the two-stage activity assay (i.e. prephosphorylation of the membranes followed by reisolation and addition of cytosol), we have shown that the major target of an activating phosphorylation is in the plasma membrane. Notably, PKCbeta, which showed the lowest EC in our system, is the major membrane translocating form of PKC in human neutrophils(42, 43) , consistent with a plasma membrane target. The particular protein target of phosphorylation remains an area for future study. A fairly large number of proteins in the plasma membrane became thiophosphorylated with [S]ATPS, complicating the identification of a particular target. Isozyme selectivity may be useful in this regard for narrowing the list of candidates. To date, the only documented PLD-related protein component in neutrophil plasma membranes is a Rho family small GTPase(10) , possibly RhoA(11) . In this regard, studies in several systems have pointed to synergy between phorbol ester and guanine nucleotide activation(27, 54) , and we have shown that inhibition of Rho with RhoGDI or GDP inhibits phorbol ester activation, (^5)consistent with a cooperative role for these two activators. Although it is possible that Rho is a direct target of phosphorylation, it seems likely that other components (e.g. the PLD itself) that are likely to be present in the plasma membrane may be the actual target of phosphorylation. It is also possible that the PKC activation induces other changes in the plasma membrane such as changes in the phospholipid composition (e.g. phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 1,4,5-trisphosphate), and that it is these changes rather than the protein phosphorylation itself that cause activation of phospholipase D. In this regard it is interesting to note recent studies of phospholipase D in U937 cells, in which phosphatidylinositol 4,5-bisphosphate was implicated as a regulator of activity(55) . It will be important in the future to isolate and reconstitute the individual PLD-related components and to investigate their regulation by phosphorylation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA46508. 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.: 404-727-5875; Fax: 404-727-2738.

(^1)
The abbreviations used are: PLD, phospholipase D; PKC, protein kinase C; DG, diacylglycerol; GTPS, guanosine 5`-O-(3-thiotriphosphate); ATPS, adenosine 5`-O-(3-thiotriphosphate); AppNHp, adenyl-5`-yl imidodiphosphate; PDBu, phorbol 12,13-dibutyrate; PMA, 4alpha-phorbol 12-myristate 13-acetate; PEth, phosphatidylethanol; TBS, Tris-buffered saline.

(^2)
During the short pretreatment time, we noted little or no additional PA or DG formed. According to previously published kinetics (7), only a small percentage of the total labeled phosphatidylcholine pool would have been metabolized within this 5-min time period, and this would be seen only when the PKC-depleted cytosol was present, since there is no PLD activity seen with PKC treatment of plasma membranes only. Therefore, substrate depletion by preactivation of PLD did not occur to an extent that might have interfered with the subsequent PLD incubation.

(^3)
I. Lopez, unpublished results.

(^4)
L. C. McPhail, unpublished results.

(^5)
I. Lopez and E. P. Bowman, unpublished results.


ACKNOWLEDGEMENTS

We thank Timothy Esbenshade for help with some of the PKC isoform experiments. We also thank Hilary Hayes for secretarial assistance.


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