©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Phospholipase D by Protein Kinase C Is Synergistic with ADP-ribosylation Factor and Independent of Protein Kinase Activity (*)

(Received for publication, November 9, 1995)

William D. Singer H. Alex Brown Xuejun Jiang Paul C. Sternweis (§)

From the Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C.(1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity.

Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKCalpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.


INTRODUCTION

Hydrolysis of phospholipids by phospholipase D (PLD) (^1)yields phosphatidic acid and the respective head groups. Phosphatidic acid is an apparent second messenger for multiple signaling events, and its production can be stimulated by a variety of stimuli, including hormones that activate G protein-mediated pathways and growth factors that function through tyrosine kinases. The phosphatidic acid produced may act directly in downstream functions or serve as a precursor for the production of diacylglycerol or lysophosphatidic acid and their subsequent sequelae. (For more recent reviews, see (1, 2, 3) .)

The stimulation of PLD in intact cells can be readily observed through measurement of the production of phosphatidic acid or phosphatidyl alcohols. However, molecular details of the mechanisms that effect this regulation are just beginning to emerge. Experiments which used permeabilized and broken cell preparations and assays which utilized exogenous lipid for substrate have elucidated at least three unique regulators of PLD activity. The first of these was revealed from the development of an assay which could use an exogenous lipid substrate to measure regulation of a PLD activity by guanine nucleotides. The activity showed a marked dependence on the inclusion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the phospholipid vesicles which contained the labeled substrate, phosphatidylcholine(4) . This unique requirement was confirmed with another system in which the utility of employing phosphatidylinositol 3,4,5-trisphosphate to measure PLD activity was also demonstrated(5) . While the molecular action of PIP(2) has not yet been determined, its requirement for the measurement of PLD that has been substantially purified (6) suggests direct action as a cofactor or regulator. A second PLD activity that can be detected with an assay that uses a mixture of phosphatidylcholine and oleate (7) does not appear to be regulated by guanine nucleotides and can be separated from the PIP(2) and G protein-dependent activity(6, 8) .

Phospholipase D activity, measured either in membranes or in a partially purified form can be stimulated by cytosolic factors. Indeed, an enriched form of PLD activity is essentially dependent on these factors(6) . The first of these factors to be purified and identified was Arf. It was purified from brain cytosol as a factor which either conferred guanine nucleotide sensitivity on a partially isolated PLD activity (4) or could reconstitute guanine nucleotide stimulated PLD activity in permeabilized cells(9) . The Arf proteins are GTP-binding proteins that occupy a unique niche in the Ras superfamily of small G proteins(10) . All of the Arf proteins tested to date are effective activators of PLD activity(6, 8) . The first Arf protein was identified as a factor which facilitated ADP-ribosylation (hence Arf) of G(s) proteins by cholera toxin(11) . More recently, Arf has been identified as a functional component of pathways for protein traffic in cells; specifically, it is a cytosolic protein required for binding of coatomer and formation of coated vesicles from Golgi membranes (see (12) for review). The speculation that regulation of PLD activity may also be important in protein transport is supported by the observations that PLD activity is higher in membranes enriched in Golgi (13) . Further, basal PLD activity appeared to be constitutively active and no longer sensitive to Arf in Golgi membranes obtained from cells resistant to the fungal metabolite, brefeldin A, an inhibitor of protein transport through Golgi.

A second group of cytosolic proteins, which have been identified as regulators of PLD activity, are members of the Rho family of monomeric G proteins. Experiments with membranes derived from neutrophils showed that GTPS stimulation of PLD activity could be attenuated by treatment with a Rho guanine nucleotide-dissociation inhibitor (GDI) protein(14) . Exton and colleagues (15) demonstrated that treatment of plasma membranes from rat liver with Rho GDI reduced guanine nucleotide-stimulated PLD activity; this activity could be restored with a purified RhoA protein. Singer et al.(16) demonstrated more directly that two purified recombinant proteins from the Rho subfamily, RhoA and Cdc42, could stimulate an enriched preparation of PLD and that this stimulation was synergistic with activation induced by Arf. The synergism observed in vitro among the three regulatory mechanisms (PIP(2), Arf, and Rho) may suggest some cooperative regulation of PLD activity by multiple pathways in vivo.

Another identified pathway for regulation of PLD activity is through protein kinase C (PKC). The PKC family has numerous members that have been studied extensively (for reviews, see (17) and (18) ). These enzymes are currently divided into three subgroups. The classical proteins (cPKC), consisting of the alpha, beta, and isoforms, are stimulated by Ca, diacylglycerol, and phosphatidylserine. The new isoforms (nPKC; , , , , µ) are not regulated by Ca and the atypical members of the family (aPKC; and ) appear to be regulated by second messengers other than diacylglycerol. Phorbol esters, which mimic diacylglycerol and activate PKC, have been used in numerous experimental paradigms which utilize intact cells to show stimulation of PLD activity. Similarly, down-regulation of PKC in various cells has led to attenuation of hormone-regulated PLD activity and thus suggested a functional role for these enzymes (see Refs. 1, 2, and 19 for reviews of earlier work).

Recent experiments by Lambeth and colleagues (20) have demonstrated that the addition of classical forms of PKC, but not the new or atypical forms, can stimulate PLD activity in membranes from neutrophils in an ATP-dependent fashion. The addition of the classical forms of PKC to Chinese hamster lung fibroblast membranes could also evoke a stimulation of PLD activity; in this case, the stimulation could be obtained in the apparent absence of ATP(21) . We have previously reported resolution of a cytosolic factor that could stimulate the activity of partially purified PLD in the absence of any nucleotide(16) . We have purified this factor and now identify it as PKCalpha. Like the resolved factor, purified PKCalpha and the recombinant protein (rPKCalpha) stimulate PLD activity in the absence of nucleotides. This action of PKC is synergistic with stimulation evoked by either Arf or RhoA, requires the presence of an activator such as Ca or phorbol 12-myristate 13-acetate (PMA), and most interestingly, is totally independent of protein kinase activity.


EXPERIMENTAL PROCEDURES

General Methods

Protein concentrations were determined by the method of Bradford(22) , using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to the method of Laemmli(23) . Concentration of proteins was performed by pressure filtration through Amicon PM 30 membranes. Solutions contained protease inhibitors when indicated at the following concentrations: 21 µg/ml N-p-tosyl-L-lysine chloromethyl ketone (TLCK), 21 µg/ml tosylphenylalanyl chloromethyl ketone (TPCK), 21 µg/ml phenylmethylsulfonyl fluoride (PMSF), 2.5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 21 µg/ml N-p-tosyl-L-arginine methyl ester.

Purification of PKCalpha from Porcine Brain Cytosol

A mixture of cytosolic activators of PLD, devoid of Arf, was obtained by DEAE-Sephacel and Ultrogel AcA 44 chromatography as described previously(16) . This preparation (approximately 1.5 g of protein in 600 ml) was brought to 2.5 mM MgCl(2), 5 mM potassium phosphate, pH 7.5, and 5 µM CaCl(2) and loaded onto a 400-ml column of hydroxylapatite (Macro Prep ceramic type I, Bio-Rad) equilibrated with Solution A (20 mM NaHepes, pH 7.5, 1 mM dithiothreitol (DTT), 50 mM NaCl, and 5 mM potassium phosphate, pH 7.5). Bound protein was eluted with a 1.8-liter linear gradient of 5-400 mM potassium phosphate, pH 7.5, in Solution A. A peak of PLD stimulatory activity devoid of RhoA immunoreactivity eluting approximately 800-1200 ml into the gradient was combined with protease inhibitors and concentrated to 20 ml. The concentrate was diluted to 200 ml with Solution B (20 mM NaHepes, pH 7.5, 1 mM EDTA, and 1 mM DTT) containing 50 mM NaCl to reduce the phosphate concentration and reconcentrated to 20 ml.

The concentrated pool of activity was diluted 2-fold with Solution B containing 4000 mM NaCl and loaded onto a fast protein liquid chromatography phenyl-Superose HR10/10 column (Pharmacia Biotech Inc.) which had been equilibrated with Solution B (2000 mM NaCl). The column was washed with 10 ml of equilibration buffer, and bound protein was eluted at 0.5 ml/min with a 90-ml linear descending gradient of NaCl (2000-0 mM) in Solution B followed by a 30-ml isocratic step without added NaCl. Stimulatory fractions (36 ml centered around 400 mM NaCl) were combined with protease inhibitors and concentrated to 12 ml. The concentrate was diluted with 8 ml of Solution B containing 2000 mM NaCl and subjected to a second step of phenyl-Superose chromatography, with elution facilitated by a 60-ml descending gradient of NaCl (1000-0 mM) in Solution B followed by a 20-ml isocratic step without added NaCl. Activity eluted at the same salt concentration as before; these factions were combined with protease inhibitors and concentrated to 0.5 ml.

The concentrated protein was diluted to 5 ml with Solution B and loaded onto a 5-ml Hi-Trap heparin column (Pharmacia). Bound protein was eluted at 1 ml/min with a two-step gradient of 0-750 mM NaCl (15 ml), then 750-2000 mM NaCl (10 ml) in Solution B. Fractions which contained the purified 80-kDa stimulatory factor (PKCalpha) were identified by SDS-PAGE and silver staining and stored at -80 °C.

Purification of rPKCalpha

Cultures (4 liters) of Spodoptera frugiperda (Sf9) cells were infected for 48 h with baculovirus directing the expression of lepine PKCalpha following established procedures(24) . The cells were harvested by centrifugation at 700 times g for 20 min, resuspended in 120 ml of lysis buffer (10 mM Tris-Cl, pH 8, 5 mM EGTA, 1 mM DTT, and protease inhibitors), and lysed by three consecutive cycles of freezing in liquid N(2) and thawing at 37 °C. The lysate was brought to 5 mM MgCl(2) and 20 µg/ml DNase A and allowed to incubate on ice for 30 min. Particulate material was subsequently removed by centrifugation at 100,000 times g for 60 min. Recombinant PKCalpha was purified from the supernatant by consecutive steps of DEAE-Sephacel, ceramic hydroxylapatite, phenyl-Superose, and Mono Q chromatography as described by Fujise et al.(25) . A final Hi-Trap heparin step was performed as for native PKCalpha to purify the protein to homogeneity. The final preparation was concentrated 10-fold, adjusted to 10% (v/v) glycerol, and stored at -80 °C.

Preparation of PLD, Arf, and RhoA

Membranes and cytosol from porcine brain were prepared as described previously(6) . Phospholipase D was extracted from the membranes with sodium cholate, exchanged into n-octyl-beta-D-glucopyranoside, and purified approximately 100-fold by a procedure concluding with Mono Q anion-exchange chromatography(6) . A highly enriched preparation of Arf (approximately 80% pure) was obtained from the cytosol after five steps of chromatography ending with passage through a Mono Q column (4) .

A DNA construct encoding glutathione S-transferase (GST) fused through a thrombin cleavage site to human RhoA with four additional amino acids at its amino terminus (Ile-Leu-Glu-Ser) was assembled in a baculovirus transfer vector derived from the pGEX-KG plasmid(26) . The construct was inserted into baculovirus by recombination and used to direct expression of protein in Sf9 cells as described elsewhere(24) . The cells were lysed by N(2) cavitation in Solution C (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM DTT, and 10 µM GDP) containing PMSF/TPCK/TLCK, and the lysate was cleared of particulate material by centrifugation at 100,000 times g for 60 min. Recombinant GST-RhoA was purified from the supernatant by chromatography through glutathione-Sepharose (Pharmacia) following standard procedures. The protein was cleaved with thrombin (1:600 thrombin:GST-RhoA by weight) for 24 h at 4 °C in Solution C containing 1% n-octyl-beta-D-glucopyranoside and 2.5 mM CaCl(2); free GST and uncleaved GST-RhoA were removed by a second passage through glutathione resin.

Treatment with Phosphatase

Recombinant PKCalpha was dephosphorylated by a modification of the procedure described by Dutil et al.(27) . The protein (0.36 µM) was incubated with varying concentrations of the catalytic subunit of protein phosphatase 1 (Calbiochem) or bovine cardiac protein phosphatase 2A (see (28) for purification) in a solution containing 20 mM NaHepes, pH 7.5, 1 mM DTT, 0.5 mM MnCl(2), 0.5 mM CaCl(2), and a sonicated dispersion of 140 µM phosphatidylserine (PS) and 4 µM dioleoylglycerol (DOG). Following incubation for 60 min at 22 °C, the reactions were quenched by the addition of 3 µM okadaic acid. Control incubations were performed with the same procedure and components except that rPKCalpha or phosphatase was omitted.

Trypsinization of rPKCalpha and Separation of Its Regulatory and Catalytic Domains

Recombinant PKCalpha (1.3 µM) was incubated with 1.2 µg/ml trypsin (TPCK-treated, Sigma) in digestion solution (20 mM Tris-Cl, pH 7.5, 1 mM DTT, 0.5 mM CaCl(2), and 10% (v/v) glycerol) for 60 min at 20 °C. Digestion was stopped by the addition of 0.1 volume of digestion solution containing 6.7 mg/ml soybean trypsin inhibitor and PMSF/TPCK/TLCK.

Separation of a micelle-bound regulatory domain from the catalytic domain was performed by a modification of a procedure described by Lee and Bell(29) . Triton X-100 (1.5% w/v) mixed micelles containing 15 mol % PS and 5 mol % DOG were diluted 10-fold into the quenched digest, and the resultant solution was incubated at 20 °C for 10 min. A 100-µl aliquot of the solution was applied to a column (9 times 0.7 cm) of Ultrogel AcA 44 resin and eluted with digestion solution containing 100 mM NaCl and 0.02% (w/v) Triton X-100. Fractions of 120 µl were collected.

Measurement of PLD and PKC Activity

PLD activity was measured by the release of [^3H]choline from exogenous phospholipid vesicles as described by Brown et al.(6) with minor modifications. Briefly, assays were performed at 37 °C in a 30-µl volume, added CaCl(2) was 3 mM (with 3 mM EGTA), and the vesicles contained 100 µM phosphatidylethanolamine, 5 µM PIP(2), and 10 µM [^3H]phosphatidylcholine. Any variations are noted in the figure legends. Analysis of phosphatidic acid production by PLD activity was performed essentially as previously described(6) , except the assays contained 3 mM CaCl(2), the vesicles consisted of a mixture of 100 µM phosphatidylethanolamine, 5 µM PIP(2), 10 µM [^14C]palmitoyl, arachidonylphosphatidylcholine, and 5 µM dipalmitoylphosphatidylcholine, and the developing solvent consisted of chloroform/methanol/acetic acid/acetone/water (10:2:2:4:1 by volume) (30) .

The activity of protein kinase C was measured by the phosphorylation of a synthetic peptide substrate (amino acids 4-14 of myelin basic protein (Calbiochem)) using a modification of a Whatman P-81 ion exchange paper binding assay described previously(31) . Reactions were performed in a 30-µl volume at 37 °C in 20 mM Tris-Cl, pH 7.5, 5 mM MgCl(2), 10 µM [-P]ATP (2-5 times 10^5 cpm), 25 µM substrate peptide, 0.2 µM CaCl(2), and 0.1% (w/v) Triton X-100 mixed micelles containing 15 mol % PS and 5 mol % DOG, and subsequently terminated by pipetting a 15-µl aliquot onto P-81 paper. Where indicated, CaCl(2), PS, and DOG were omitted from the assay mixture. In some instances, the phospholipid vesicles described for the PLD assay were substituted for Triton X-100 mixed micelles.


RESULTS

Purification and Identification of PKCalpha as a Cytosolic Activator of PLD

A cytosolic factor capable of activating PLD in the absence of any nucleotide was purified from porcine brain cytosol. The unidentified factor was resolved with RhoA from Arf by consecutive steps of DEAE and AcA 44 chromatography(16) . A third chromatographic step through hydroxylapatite produced two resolved peaks of PLD stimulatory activity (see ``Experimental Procedures'' for details of purification). While both peaks were capable of activating PLD in the absence of added nucleotides, the earlier eluting peak contained RhoA immunoreactivity and could stimulate better with guanine nucleotides. The nucleotide-independent stimulatory factor was purified from the later peak by three more chromatographic steps and eluted as a polypeptide migrating through SDS-gels with an apparent molecular mass of 80 kDa (Fig. 1). Amino acid sequences of peptides obtained from Lys-C digestion of the purified factor were an exact match with sequences from rat brain PKCalpha (data not shown). Recognition of the 80-kDa protein by immunoblot analysis with antibodies specific for PKCalpha further confirmed this identity.


Figure 1: Purified cytosolic factor and rPKCalpha. The cytosolic factor which stimulated PLD in a nucleotide-independent fashion and rPKCalpha were purified as described under ``Experimental Procedures.'' Samples (50 ng) of each were resolved by SDS-PAGE on a 12% acrylamide gel. Proteins were visualized by staining with silver.



The yield of purified PKCalpha from porcine brain (approximately 150 µg from 30 g of cytosolic protein) was insufficient for extensive characterization of its interaction with PLD and other cytosolic factors. Therefore, recombinant protein was produced with a baculovirus expression system(25) . As shown in Fig. 1, a homogenous preparation of rPKCalpha was obtained from the cytosol of infected cells after five steps of chromatography (see ``Experimental Procedures'') and a yield of 0.5 mg/liter of culture. The stimulation of PLD activity using the recombinant protein was similar to that of native porcine PKCalpha.

Activation of PLD by rPKCalpha

The dependence of the activation of PLD on the concentration of rPKCalpha is shown in Fig. 2. The partially purified PLD used for these studies was devoid of immunoreactivity for Arf, RhoA, and PKCalpha and contained no detectable GTPS-binding activity (data not shown). Under all conditions there was a potent and robust concentration-dependent stimulation of PLD activity that was half-maximal at 20-30 nM and maximal at 100 nM rPKCalpha. A decrease in activity was observed at higher concentrations (>200 nM) of the enzyme. The efficacy of rPKCalpha was essentially identical in the absence or presence of ATP or GTPS. This indicates that the kinase activity of the PKC is not required for activation of PLD. A small reproducible increase in activation by rPKCalpha was observed consistently in the presence of both ATP and GTPS; the reason for this is not known at present.


Figure 2: Activation of PLD by rPKCalpha. Assays of PLD activity were conducted for 30 min as described under ``Experimental Procedures.'' Assays contained 0.9 µg of PLD, the indicated nucleotide (10 µM), and variable amounts of rPKCalpha. Activities shown for the lowest concentration of rPKCalpha in the figure were essentially the same as obtained in the absence of rPKCalpha.



Interactions among rPKCalpha, Arf, and RhoA

Fig. 3demonstrates the pronounced synergistic stimulation of PLD that occurred in the presence of both PKCalpha and Arf. Titration of the factors against fixed amounts of each other revealed clear synergism (3-4-fold) at both suboptimal and saturating concentrations. The latter indicates that PKC and Arf increase the actual efficacy of each other. A small increase in potency induced by either factor on the other also contributes to the synergism.


Figure 3: Dependence of the activation of PLD on the concentration of rPKCalpha and Arf. Assays for enzyme activity were conducted for 7.5 min as described under ``Experimental Procedures.'' Assays contained 10 µM GTPS, 10 µM ATP, 0.9 µg of PLD, and the indicated fixed or variable concentrations of rPKCalpha and Arf. Activities shown for the lowest concentration in the titrations were essentially the same as for no addition of the titrated factor.



Routine assays of PLD activity measure the release of water-soluble choline from phosphatidylcholine. The lipid products of phosphatidylcholine hydrolysis in the presence of PKC and Arf were also analyzed to ensure that the synergism elicited by these two proteins reflects only the stimulation of PLD activity, not a stimulation of PLD in conjunction with another lipase. Phosphatidic acid was the only detectable product of phosphatidylcholine hydrolysis stimulated by PKC and Arf either alone or in combination (summarized in Table 1). Production of phosphatidyl alcohol and concomitant reduction in phosphatidic acid formation occurred in the presence of ethanol, as expected for the transphosphatidylation reaction characteristic of PLD enzymes (for review, see (32) ).



Synergism was also obtained when PLD was stimulated with either rPKCalpha or Arf in the presence of recombinant RhoA (rRhoA) (Fig. 4). The rRhoA required preactivation with GTPS to be effective in this assay. While the RhoA produced little stimulation by itself, it enhanced the activity of the other two factors 3-4-fold. A similar increase occurred for the combination of PKCalpha and Arf. Interestingly, the synergistic action was higher (6.3-fold) when all three factors were employed together.


Figure 4: Activation of PLD by rRhoA, Arf, and rPKCalpha. Phospholipase D activity was determined as described under ``Experimental Procedures.'' Assays were conducted for 7.5 min and contained 0.9 µg of PLD, 10 µM GTPS, 10 µM ATP, and combinations of 1 µM rRhoA, 20 nM Arf, and 10 nM rPKCalpha as indicated.



Effect of Activators and Inhibitors of PKC on Activation of PLD

Table 2shows the results of several well defined modulators of PKC on its ability to activate PLD. Several points emerge. First, activation of the PKC is required. In the absence of Ca, PLD was not activated by PKCalpha. Addition of 3 mM Ca (10 µM calculated free Ca) allowed stimulation by the factor (PKCalpha) alone and synergism with Arf. As reported previously(6) , Ca produced only a small increase in stimulation of PLD by Arf alone. Inclusion of 1 µM PMA promoted activation of PLD by PKC in the absence of Ca and had no affect on stimulation by Arf.



The modulators described above activate PKC through interactions with its regulatory (lipid-binding) domain. Staurosporine inhibits the kinase activity of the proteolytically generated catalytic fragment of PKC while having no effect on the binding of phorbol esters to the regulatory domain(33) . Concentrations of staurosporine which inhibited 77% of the kinase activity of PKCalpha had little effect on activation of PLD by the enzyme or its synergism with Arf (Table 2, part B).

Effect of Dephosphorylation of rPKCalpha on PLD Activation

Removal of a permissive phosphorylation on PKC by phosphatase treatment is reported to eliminate its kinase activity (27) . The effects of incubation of PKC with the catalytic subunit of protein phosphatases 1 or 2A on stimulation of PLD and kinase activity are shown in Fig. 5. Increasing concentrations of phosphatase resulted in an increasing shift from an apparent molecular mass of 80 kDa to 76 kDa on SDS-gels (top panels), indicative of the removal of the permissive phosphorylation(27) . Dephosphorylation by protein phosphatase 1 appeared to be more efficient than protein phosphatase 2A when compared at the same concentration. The reduction in permissive phosphorylation correlated with a decrease in the ability of PKC to phosphorylate substrate peptide (bottom panels). In contrast, there was little difference in the ability of phosphorylated and dephosphorylated PKC to activate PLD. Activation was measured without added nucleotide to eliminate the possibility of rephosphorylation of rPKCalpha by a contaminating kinase. Synergistic activation with Arf in the presence of 1 µM GTPS was also relatively unaffected by dephosphorylation. Experiments where protein phosphatase 1 or 2A were treated with okadaic acid and then added with PKC in assays for kinase or PLD activity yielded similar results to those obtained in the absence of phosphatase (data not shown).


Figure 5: Effect of dephosphorylation of rPKCalpha by protein phosphatases 1 (PP1) and 2A (PP2A) on its kinase activity and ability to activate PLD. Dephosphorylation reactions were performed with the indicated concentrations of protein phosphatase catalytic subunits as described under ``Experimental Procedures.'' Top, samples of phosphatase-treated rPKCalpha (75 ng) were resolved by SDS-PAGE on 9% acrylamide gels and visualized by staining with silver. Bottom panels, assays for PLD and kinase activity were performed for 30 min and 15 min, respectively, as described under ``Experimental Procedures.'' Data are expressed as percent activity of phosphatase-treated rPKCalpha relative to rPKCalpha treated with the same buffer but without the phosphatase. The PLD assays contained 0.9 µg of PLD, 30 nM rPKCalpha, 1.25 µM okadaic acid, and 1 µM GTPS in the presence of 100 nM Arf or no added nucleotides in the absence of Arf. The amount of phosphatidylcholine (PC) hydrolyzed by untreated rPKC was 8 pmol without Arf and 83 pmol with Arf. The phosphorylation of substrate peptide was monitored in the presence of PS, DOG, Ca, 1.25 µM okadaic acid, and 30 nM rPKCalpha. Untreated rPKCalpha phosphorylated 85 pmol of peptide.



Evaluation of Activation of PLD by Regulatory and Catalytic Domains of PKCalpha

The classical forms of PKC can be cleaved by limited proteolysis into two domains, an amino-terminal lipid-binding regulatory domain and a carboxyl-terminal kinase domain. These domains can then be physically separated into individual functional entities (29) . Such a digestion of rPKCalpha with trypsin and separation of its fragments is shown in Fig. 6.


Figure 6: Separation of the regulatory and catalytic domains of trypsin-treated rPKCalpha by gel filtration. Recombinant PKCalpha was treated with trypsin and subjected to chromatography through Ultrogel AcA 44 as described under ``Experimental Procedures.'' Top panel, aliquots (19 µl) of the fractions were resolved by SDS-PAGE on 12% acrylamide gels and polypeptides were visualized by silver staining. Lanes designated rPKC and Digest contained 125 ng of the protein before and after proteolysis, respectively. Bottom panel, aliquots (2.5 µl) of the fractions were analyzed for their ability to activate PLD or phosphorylate substrate peptide as described under ``Experimental Procedures.'' Assays for PLD were conducted for 30 min and contained 0.9 µg of PLD, 10 µM GTPS, and 10 µM ATP in the presence or absence of 100 nM Arf. Phosphorylation of substrate peptide was assessed after a 15 min incubation in the presence or absence of PS, DOG, and Ca.



Analysis by SDS-PAGE (top panel) indicated that digestion of the 80-kDa rPKCalpha (rPKC lane) resulted in almost complete conversion (Digest lane) into fragments which included those of 50-52 kDa, the reported size (29) of the kinase domain, and 34 kDa, the size of the regulatory domain. An antibody raised against a peptide corresponding to the 12 carboxyl-terminal amino acids of PKCalpha recognized only the 50-52-kDa fragments (data not shown). Additional fragments of 40-43 kDa were also produced which probably represent further degradation of the catalytic domain; they did not contain the lipid-binding domain, based on their inability to bind to mixed micelles of Triton X-100 and lipid (see below). The 21-kDa protein in the rPKC and Digest lanes is soybean trypsin inhibitor; staining in the 66-kDa region is an artifact. Proteolysis had only a minor effect on the ability of PKC to activate PLD; undigested PKC (30 nM) in the presence of Ca stimulated 86 and 26 pmol of phosphatidylcholine hydrolysis with and without 100 nM Arf, respectively. This compares with stimulated hydrolysis of 102 and 21 pmol of phosphatidylcholine after digestion.

When mixed micelles of Triton X-100, PS, and DOG were added to trypsinized PKCalpha and the resulting solution was applied to an AcA 44 size exclusion column, the elution pattern shown in the top panel of Fig. 6was obtained. The majority of the 34-kDa fragment and a detectable residue of undigested PKC eluted well before the 50-52- and 40-43-kDa pieces. The larger size of the regulatory domain is expected from its association with the mixed lipid/detergent micelles. Stimulatory activity for phospholipase D (bottom panel) coeluted with the 34-kDa fragment; this indicates that the regulatory domain alone may be sufficient to provide stimulation of PLD by PKC. A peak of protein kinase activity eluted well after the major portion of PLD stimulatory activity, and overlapped almost exactly with the 50-52-kDa fragments. This kinase activity is not dependent on Ca or phospholipids, as expected for removal of the regulatory domain. It is apparent that the isolated kinase domain is not an effective activator of PLD, even when assayed in the presence of 10 µM ATP.


DISCUSSION

PKCalpha was identified as a cytosolic factor from porcine brain capable of stimulating PLD activity in the absence of added nucleotides. This action of the enzyme through a molecular mechanism which did not involve its kinase activity was supported by three other experimental findings: 1) staurosporine significantly inhibited kinase activity at a concentration which did not inhibit activation of PLD, 2) dephosphorylated (kinase-deficient) PKC stimulated PLD, and 3) the isolated active kinase domain produced by trypsin treatment of the enzyme did not stimulate activity, whereas the regulatory domain was an effective activator. This robust phosphorylation-independent stimulation by PKCalpha was observed with a PLD activity that had been extracted from porcine brain membranes, was purified away from other known regulators such as Arf and RhoA, and which displayed essentially no basal activity. This nucleotide independence of PKCalpha action concurs with the report of a 2-3-fold stimulation of PLD activity in the absence of added ATP following addition of the classical isotypes of PKC to a crude membrane preparation from Chinese hamster lung fibroblasts(21) . In contrast, stimulation of PLD activity in human neutrophil membranes was reported to require phosphorylation of a target protein by added cPKCs(20) . The latter observations may reflect the regulation of a different isotype of PLD or an alternative pathway in neutrophil membranes for activation of the same PLD.

The phosphorylation-independent mechanism for activation of PLD by PKCalpha remains unidentified. Here, as in other studies(20, 34) , stimulation of PLD required activators of PKC (e.g. Ca or PMA). Low micromolar concentrations of PIP(2) were also needed for activation (data not shown), although the general requirement of this lipid for PLD activity makes it unclear whether this effect involves a direct interaction between PIP(2) and PKC. It should be noted that the PIP(2)-containing vesicles used in the PLD assay promoted activation of PKC as assessed by stimulation of its kinase activity (data not shown). Mixed micelles of detergent and PIP(2) have also been reported to stimulate the kinase activity of PKC(35) , presumably reflecting the interaction of anionic lipids with its lipid-binding domain. Current models predict that activators of PKC promote a molecular transformation that disinhibits the kinase active site of the enzyme and exposes membrane association sites in its regulatory domain. Presumably, this same molecular transformation induces an interaction site for stimulation of PLD. Binding of PKC to an anionic phospholipid surface could facilitate this activation of PLD through increased protein-protein interaction. In the absence of pure PLD it is not clear whether this regulation involves a direct complex of PKC with PLD, or an interaction between PKC and a protein which in turn affects PLD.

Protein kinase C not only stimulates PLD activity, but also enhances the interaction of PLD with other activators. Clear synergism of PKC with Arf or RhoA was observed with the partially purified PLD. This corresponds with earlier reports of the potentiation of GTPS-stimulated PLD by phorbol ester in permeabilized cells and cell-free systems (reviewed in(3) ). Cooperative stimulation of PLD activity was also observed when RhoA and PKC were added to HL60 membranes which had been pretreated with Rho GDI to remove endogenous Rho proteins(36) . Arf has been shown to act synergistically with members of the Rho family(16, 37, 38) , and also with a ``50-kDa'' fraction prepared by gel filtration of cytosol from HL-60 cells (39) and human neutrophils (40) which contains members of the Rho family and at least one other unidentified activator. It should be noted that RhoA and the nucleotide-independent factor identified here as PKCalpha coeluted during gel filtration of a cytosolic fraction from porcine brain(16) .

Inactivation of the kinase activity of PKCalpha by dephosphorylation had no effect on its synergistic activation of PLD with Arf. Therefore, this action also arises through a phosphorylation-independent mechanism. Since both PKC and Arf alone activate PLD, their synergism probably involves a cooperative interaction through different sites on the enzyme. Interestingly, treatment of rat basophils with phorbol esters is reported to promote Arf binding to the Golgi membrane(41) . Treatment of HL-60 cells with PMA or fMet-Leu-Phe promotes translocation of Arf to membranes and correlates with increased stimulation of PLD by GTPS(42) . It is unclear how factors that promote PKC translocation to membranes in turn promote Arf translocation, but it is possible that membrane-associated PKC serves as a nucleating agent for the formation of an active PLD complex that includes other activators such as Arf and RhoA. It seems probable that the most efficacious regulation of PLD may occur through the formation of highly cooperative complexes which are facilitated by the merger of a variety of signal transduction pathways.

The coelution of PLD stimulatory activity with the regulatory domain, and not the kinase domain, during gel filtration of PKC treated with trypsin, indicates that this lipid-binding portion of PKCalpha contains a site for complexation with PLD and/or related proteins. While it is possible that a small amount of uncleaved PKC detected with the regulatory domain contributed to this activity, it cannot account for the total activity observed; fractions of the regulatory domain were estimated to contribute less than 0.5 nM uncut PKC to the PLD assays, and activation or synergism with Arf has never been detected at subnanomolar concentrations of PKC (see Fig. 2and Fig. 3).

Overall, these studies have important implications for the function of PKC in the cell. Following an agonist-induced increase in cytosolic free Ca and diacylglycerol (e.g. through activation of phospholipase C), PKCalpha translocates to the cell membrane where it is tethered in an active form by lipids (diacylglycerol, PS, and PIP(2)) and receptor proteins, such as receptors for activated protein kinase C (RACKs)(43) . Here, it may effect signal transduction in a bimodal manner, through phosphorylation of substrates and, as demonstrated here for PLD, by direct protein-protein interaction. It will be of interest to determine whether this latter action of PKC is operative on other downstream effectors of the kinase and whether this dual mode of regulation may be a functional mechanism used by other protein kinases.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant GM31954, National Service Research Award GM15817 (to H. A. B.), and the Robert A. Welch Foundation. 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: Dept. of Pharmacology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-2835; Fax: 214-648-2971.

(^1)
The abbreviations used are: PLD, phopholipase D; GTPS, guanosine 5`-O-(3-thiotriphosphate); PIP(2), phosphatidylinositol 4,5-bisphosphate; Arf, ADP-ribosylation factor; GDI, guanine nucleotide-dissociation inhibitor; PKC, protein kinase C; rPKCalpha, recombinant alpha isoform of protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; TPCK, tosylphenylalanyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; GST, glutathione S-transferase; PS, phosphatidylserine; DOG, dioleoylglycerol; rRhoA, recombinant RhoA.


ACKNOWLEDGEMENTS

We thank John Long and Steve Gutowski for their excellent technical assistance and Kim Edwards for administrative support. We also thank Tohru Kozasa and Shigeo Ohno for the provision of PKCalpha baculovirus, Gary Bokoch for RhoA cDNA, and Craig Kamibayashi and Marc Mumby for purified protein phosphatase 2A catalytic subunit. Finally, we thank Steve Afendis, Carolyn Moomaw, and Clive Slaughter for sequence analysis of PKCalpha.


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