©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Resolved Phospholipase D Activity Is Modulated by Cytosolic Factors Other than Arf (*)

William D. Singer (1), H. Alex Brown (1), Gary M. Bokoch (2), Paul C. Sternweis (1)(§)

From the (1)Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9041 and the (2)Department of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D, which has been extracted from porcine brain membranes and chromatographically enriched 100-fold, was activated better by impure preparations of Arf than by purified or recombinant Arf. Examination of brain cytosol with this enriched preparation of PLD activity revealed at least three stimulatory components. One of these is Arf or the first cytoplasmic factor. A second peak of PLD-stimulating activity (cytoplasmic factor II, CFII) was resolved from Arf by anion exchange and gel filtration. This CFII can be further separated into multiple activities by chromatography with heparin-agarose. The activities were differentiated by their stimulatory properties as measured in the absence or presence of guanosine 5`-O-(3-thiotriphosphate) (GTPS) alone and in the presence of added Arf and GTPS.

While all of the CFII pools stimulated PLD activity to some degree and showed synergistic activation when administered in conjunction with Arf, they could be classified into two groups with distinct behavior. When used together, pools from the two respective groups showed synergistic activation of PLD. The first set of pools contained the RhoA monomeric G protein. Recombinant RhoA was used to show that it could indeed activate this enriched PLD activity and act synergistically with Arf proteins. A related monomeric G protein, Cdc42, was also effective. The second set of CFII pools were devoid of RhoA and, in contrast to the first group, demonstrated significant stimulating activity in the absence of guanine nucleotides.

These data indicate that the PLD activity from brain can be modulated by several cytosolic factors and that Arf-sensitive PLD may represent a complex activity that can be regulated in an interactive fashion by a variety of cellular signaling events.


INTRODUCTION

Phospholipase D (PLD)()hydrolyzes phospholipids to produce phosphatidic acid and their respective head groups. This activity can be regulated by a variety of hormones and growth factors and one of the products, phosphatidic acid, has been touted as a putative second messenger (1, 2) and has also been suggested to have fusigenic properties(3) . Phosphatidic acid is also a precursor of the second messenger, diacylglycerol, an activator of protein kinase C.

Characterization of mammalian PLD (4, 5, 6) has lagged behind that of the plant enzyme (for reviews, see Refs. 4-7, and see Ref. 8 for the first cloned enzyme from plants). Furthermore, the regulation of PLD activity is not well understood. Several lines of evidence indicate that GTP-binding regulatory proteins (G proteins) are involved (for review, see Ref. 6). For example, stimulation of PLD activity by a variety of receptors that are known to act through heterotrimeric G proteins indicate their involvement. Recently, two groups have demonstrated, either through in vitro assays with exogenous substrate (9) or through use of permeabilized cells(10) , that PLD activity can be stimulated by the monomeric G protein, Arf. Further evidence of involvement of the monomeric G proteins comes from the demonstration that Rho GDI, a GDP dissociation inhibitor of the Rho family of monomeric G proteins, could inhibit activation by GTPS of PLD in neutrophil membranes (11) and that recombinant RhoA could stimulate a PLD activity in rat liver plasma membranes(12) .

The previous accompanying manuscript (13) describes the partial purification and characterization of Arf-sensitive PLD activity from porcine brain. During this purification, it became apparent that there were other factors that could stimulate the same PLD activity. This report describes the initial resolution of these other cytosolic factors from Arf and initial attempts to identify the components and characterize their activities.


EXPERIMENTAL PROCEDURES

Preparation of PLD and Arf

Porcine brain membranes and cytosol were prepared as described in the accompanying report(13) . Briefly, PLD was extracted from the membranes with sodium cholate, exchanged into n-octyl--D-glucopyranoside, and partially purified through three steps of chromatography, which concluded with passage through a Mono Q anion-exchange column. The PLD in this preparation is enriched about 100-fold. A highly enriched preparation of Arf (approximately 25% pure) was obtained from porcine brain cytosol after four steps of chromatography, which terminated with gel filtration through Sephadex G-75(9) . Myristoylated recombinant human Arf5 (myrArf5) was produced by co-expression with a yeast myristoyl transferase in Escherichia coli(14) .

Preparation of CFII Factors

CFII activity is defined as the ability to stimulate PLD activity in the absence of Arf or an ability to enhance PLD activity in the presence of Arf (see ``Results'' for further description). All procedures were conducted at 4 °C. Protease inhibitors were included 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, 2.5 µg/ml leupeptin, 0.1 unit of trypsin inhibitor/ml of aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml pepstatin A, and 21 µg/ml N-p-tosyl-L-arginine methyl ester.

Porcine brain cytosol (5-6 g of protein) was diluted with an equal volume of Solution A (20 mM Tris-Cl, pH 8, 1 mM EDTA, 1 mM dithiothreitol, and 1 µM GDP) containing phenylmethylsulfonyl fluoride/TLCK/TPCK and loaded onto a 1-liter column of DEAE-Sephacel (Pharmacia Biotech Inc.), which had been equilibrated with the same solution. Bound protein was eluted with a 2-liter linear gradient of NaCl (0-300 mM) in Solution A, followed by a 1.2-liter wash with 1 M NaCl in Solution A. Fractions of 27 ml were collected, and samples were assayed for Arf activity and the capacity to stimulate PLD activity. CFII activity eluted over approximately 25 fractions centered around 110 mM NaCl (see ``Results'' for basis of CFII peak selection). Fractions with activity were combined with protease inhibitors and concentrated to 30 ml by pressure filtration through an Amicon PM 10 membrane.

The concentrated pool of activity from DEAE was loaded onto a 1.2-liter column of Ultrogel AcA 44 (Sepracor), which was both equilibrated and eluted with 1.2 liters of Solution B (20 mM NaHepes, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 1 µM GDP). Fractions of 20 ml were collected.

A portion (60 ml) of the pool of CFII activity from the AcA 44 step was loaded onto a 50-ml column of heparin-Sepharose CL-6B (Pharmacia), which had been equilibrated with Solution B containing protease inhibitors. Bound protein was eluted with a 200-ml linear gradient of NaCl (50-500 mM) in Solution B, followed by a 75-ml wash with 1.5 M NaCl in the same solution. The flow-through of applied material was collected as fractions 1-8; subsequent fractions contained 4.5 ml. Three regions of PLD stimulatory activity were observed (see Fig. 3); these were designated A, B, and C, and representative pools were collected as indicated.


Figure 3: Chromatography of CFII factors through heparin-Sepharose CL-6B. A portion (60 ml) of the pool of activity from the Ultrogel AcA 44 step was fractionated through heparin-Sepharose and assayed as described under ``Experimental Procedures.'' Assays to measure activation of PLD were the same as indicated in Fig. 1, except for the use of 0.5 µg of PLD, 1 µg of porcine Arf, and the inclusion of 2.8 mMn-octyl--D-glucopyranoside. Fractions selected for pools of activity are indicated by the linesbetweenpanels: PoolA, fractions 8-10; PoolB, fractions 25-28; and PoolC, fractions 36-42.



Each pool of activity from heparin-Sepharose was loaded onto a fast protein liquid chromatography hydroxylapatite column (100 7.8 mm Bio-Gel high performance hydroxylapatite column, Bio-Rad) equilibrated with Solution C (20 mM NaHepes, pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, 10 µM CaCl, 5 mM potassium phosphate, pH 7.5, and protease inhibitors). The column was eluted at 0.5 ml/min with a 50-ml linear gradient of 5-350 mM potassium phosphate, pH 7.5, in Solution C. Fractions of 2 ml were collected in tubes containing a 10-µl aliquot of 200 µM GDP to achieve a concentration of 1 µM GDP in the fractions. Pools of CFII activities were collected as indicated. Stimulatory fractions from B were separated into two pools, designated CFII B and CFII B`. The pools were concentrated 5-10-fold by ultrafiltration using Centricon 10 microconcentrators (Amicon) and stored in aliquots at -80 °C.

Measurement of PLD and Arf Activity

PLD activity was assessed by its ability to hydrolyze dipalmitoyl-PC using an assay described previously (9, 13) with minor modifications. Briefly, 5 µl of mixed lipid vesicles containing 3 nmol of phosphatidylethanolamine, 0.3 nmol of phosphatidylinositol 4,5-bisphosphate (PIP), 0.3 nmol of dipalmitoyl- PC, and [choline-methyl-H]dipalmitoyl-PC to yield approximately 30,000 cpm/assay were added to a mixture of PLD, cytosolic factors, and regulatory ligands (such as GTPS) in a total volume of 30 µl. The assays also contained 1.7 mMn-octyl--D-glucopyranoside (which inhibits the assay(15) ), due to the presence of the detergent in the PLD preparation. Reactions were allowed to proceed for 30 min at 37 °C. Variations from this protocol are indicated in the figure legends.

Arf activity was determined by its ability to promote ADP-ribosylation of recombinant G in the presence of activated cholera toxin. Aliquots (10 µl) of column fractions were assayed essentially as described previously(9) , except 20 pmol of bovine brain heterotrimeric G protein subunits was employed, the concentration of activated cholera toxin added to the assay was increased to 500 µg/ml, and the reaction was terminated after 60 min at 30 °C.

Immunoblot Analysis

Proteins in column fractions were resolved by electrophoresis through 12% SDS-polyacrylamide gels (16) and transferred to nitrocellulose. Following incubations with primary antibody (1 µg/ml) and horseradish peroxidase-conjugated secondary antibody (Amersham Corp.), immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Corp.). Affinity-purified antibodies raised against peptides representing portions of RhoA and Rho GDI were obtained from Santa Cruz Biotechnology.

Miscellaneous Methods

Guanine nucleotide binding activity was quantified by the filter binding method(17) . Aliquots (5 µl) of column fractions were assayed for 60 min at 30 °C in a 60-µl volume of solution containing 50 mM NaHepes, pH 8, 1 mM EDTA, 1 mM dithiothreitol, 800 mM NaCl, dimyristoyl-PC (3 mM) vesicles, 0.1% sodium cholate, 5 mM MgCl, and 1 µM [S]GTPS (4000 cpm/pmol).

Recombinant human RhoA(18) , Rac1(19) , and Cdc42 containing posttranslational modifications were purified from Spodoptera frugiperda (Sf9) cells after expression using a baculovirus system. Human Rho GDI (20) and nonmodified RhoA containing the Val-14 activation mutation (18, 21) were purified following overexpression in E. coli. Nonprenylated recombinant human Rab1a, Rab3a, and Rab5 (all containing a hexahistidine tag at their amino terminus), were also purified following expression in E. coli(22) . These Rab proteins, and the prenylated recombinant Rab1a without a hexahistidine tag, were provided by Dr. Miguel Seabra. Recombinant G, obtained by expression in E. coli and purified according to published procedures(23) , was a gift of the laboratory of Dr. Alfred Gilman. Heterotrimeric G protein subunits were purified from bovine brain as described previously(24) . Protein concentrations were determined by the method of Bradford(25) , using bovine serum albumin as a standard.


RESULTS

The original isolation of Arf as a cytosolic factor for stimulation of PLD activity was accomplished with either extracts of membranes (9) or permeabilized cells (10) as the source of the enzyme. The accompanying report (13) described the use of Arf to partially purify and characterize PLD activity from porcine brain. During the course of these studies, it became apparent that cruder preparations of Arf were more effective than highly purified and recombinant Arf in stimulating more enriched preparations of PLD. This prompted a further evaluation of cytosol in which enriched porcine PLD (purified about 100-fold from membranes) was used as the source of activity.

Resolution of Stimulatory Cytosolic Factors from Arf

The profiles of activities obtained from the first two steps of chromatographic resolution of porcine brain cytosol are shown in Figs. 1 and 2. Activities obtained after mixing enriched PLD with aliquots of each fraction were measured under two conditions; in the presence of either GTPS or added Arf and GTPS.

Separation via anion exchange (Fig. 1) revealed clear overlapping peaks of activity with both conditions. Binding of GTPS and the presence of Arf in the fractions was also assessed for comparison. Two observations are worthy of note. First, GTPS stimulation of PLD activity () did not correlate well with the presence of Arf (); that is, fractions that contain little or no Arf can stimulate PLD activity. Second, even in the presence of near saturating levels of Arf (), additional stimulating activity was observed in fractions(60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85) following the peak of Arf. These results indicated that cytosol contains stimulatory factors for PLD other than Arf.


Figure 1: Resolution of CFII activity and Arf activity by chromatography through DEAE-Sephacel. Porcine brain cytosol was fractionated and assayed as described under ``Experimental Procedures.'' Aliquots (5 µl) of fractions were examined for their capacity to stimulate PLD activity by assays that contained 0.2 µg of PLD, 10 µM GTPS, and 8.2 µg of porcine brain Arf as indicated. Fractions 61-85 were collected as the CFII pool.



To pursue these other factors, a pool (CFII) of fractions that contained stimulating activity but which were mostly devoid of Arf activity was concentrated and subjected to gel filtration (Fig. 2). Again, two assay conditions used to look at PLD-stimulating activity resulted in definable peaks of activity. GTPS alone () revealed two peaks of activity; the peak with slower migration corresponded to fractions that contained Arf. A peak (CFII) with faster migration lacked Arf but stimulated PLD activity in the presence of GTPS alone or enhanced activity in the presence of added Arf and GTPS. It is of interest to note that the stimulating peak devoid of Arf also contained GTPS binding activity.


Figure 2: Resolution of CFII factors and Arf by chromatography through Ultrogel AcA 44. The pool of activity from the DEAE-Sephacel column was fractionated and assayed as described under ``Experimental Procedures.'' Assays for PLD-stimulating activity were the same as indicated in Fig. 1. Fractions 30-38 were collected as the CFII pool (180 ml, 450 mg of protein).



Resolution of PLD-stimulating Activities on Heparin-Sepharose and Hydroxylapatite

The CFII peak of PLD stimulating activity with faster migration through gel filtration was further resolved on heparin-Sepharose (Fig. 3). Multiple activities appeared to emerge. Whereas a well defined peak of activity with the capacity to stimulate PLD in the presence of GTPS alone was observed, the capability to enhance PLD activity in the presence of Arf and GTPS was broadly distributed. ADP-ribosylation activity and Arf immunoreactivity were not detectable in the heparin fractions (data not shown). Interestingly, fractions in the latter part of the elution, which could enhance stimulation of PLD in the presence of Arf, were devoid of GTPS binding activity. Fractions were selected to represent potential resolved activities (Fig. 3). Pool A (early in the elution) contained GTPS binding activity but required Arf to be effective. Pool B could activate PLD in the absence of Arf. Pool C (later in the elution) represented a capacity to activate PLD in the presence of Arf but had little intrinsic GTPS binding activity.

The three pools were subjected to further resolution on hydroxylapatite. The profiles of activities eluted by identical chromatography of the three pools through this matrix are shown in Fig. 4. Whereas Pools A and C eluted as apparent singular peaks with uniform behavior, Pool B from the heparin-Sepharose appeared to separate into two activities. One pool (labeled B) enhanced stimulation of PLD activity in the presence of Arf but had little effect in the presence of GTPS alone; a second pool (labeled B`) stimulated PLD activity in the presence of GTPS alone as well as showing enhancement of stimulation by Arf. Another criterion for the separation of the B and B` pools was either the presence or absence, respectively, of the RhoA protein (see below). Selected fractions were pooled as indicated and concentrated for further characterization.


Figure 4: Chromatography of CFII factors through hydroxylapatite. Pools A (top panel), B (middle panel), and C (bottom panel) from the heparin-Sepharose CL-6B column were fractionated through a Bio-Gel high performance hydroxylapatite column and assayed as described under ``Experimental Procedures.'' Stimulation of PLD activity was measured as described for Fig. 3. Fractions containing activity were pooled as indicated in each panel.



Interactions among Arf and the CFII Pools

The stimulatory affect of Arf is compared with activation obtained with the CFII peaks in Fig. 5. These were all measured in the presence of GTPS. Within the ranges of concentrations that could be tested, both Arf and the CFII B` pool gave robust stimulation. The other three factors were either less efficacious or less potent. For example, the effect of CFII B was still increasing at the highest level of factor tested, and the exact nature of the activation by Peak CFII C is hard to evaluate due to an inhibitory action when high concentrations of the pool were used.


Figure 5: Activation of PLD with resolved cytosolic factors. PLD activity was determined as described under ``Experimental Procedures.'' Assays contained 10 µM GTPS, 1 µg of PLD, and the indicated amount of cytosolic factor.



The observations are of particular interest when the action of Arf was examined in the presence of the cytosolic pools (Fig. 6). Besides their own component of stimulatory action, the cytosolic factors increased the potency of Arf. This is most pronounced for the CFII B` pool, which increased the potency of Arf by approximately 10-fold. The other three factors induce a more modest effect, about a 3-fold increase in the potency of Arf. At maximal concentrations of Arf, the cytosolic factors gave no more than additive effects. Therefore, the primary result of combining the factors with Arf was a synergistic action at suboptimal concentrations of Arf. This synergism with Arf was also observed when the factors were titrated against fixed concentrations of Arf ( Fig. 6and Fig. 7).


Figure 6: Concentration dependence of Arf, CFII A, and CFII B for the activation of PLD in the presence of other cytosolic factors. Enzyme activity was determined as described under ``Experimental Procedures.'' Assays contained 10 µM GTPS, 1 µg of PLD, and either 1.2 µg of porcine brain Arf, 1 µg of CFII A, 5 µg of CFII B, 0.6 µg of CFII B`, or 1.5 µg of CFII C as indicated for titration curves. In addition, assays contained the indicated variable amounts of Arf, CFII A, or CFII B. Activities shown for the lowest concentration in the titrations were essentially the same as no addition of the titrated factor.




Figure 7: Concentration dependence of CFII B` and CFII C for the activation of PLD in the presence of other cytosolic factors. Assays were performed with the indicated amounts of factors as described in Fig. 6 and the indicated variable amounts of CFII B` or CFII C.



When the factors were mixed with each other, synergistic action could also be observed between factors CFII A and either CFII B` or CFII C and between factors CFII B and either CFII B` or CFII C (Fig. 6). Since the cytosolic factors were not sufficiently concentrated to saturate their effects on PLD, it is not clear whether the synergism is due to a shift in the potency of the factors or to an actual synergism in total efficacy.

Only additive or less than additive effects are observed when CFII A is combined with CFII B or when CFII B` is combined with CFII C ( Fig. 6and 7). The combination of this result and similarities in synergistic action suggest that CFII A and CFII B may represent the same or similar factors. Such a relationship may also exist between CFII B` and CFII C.

Fig. 8demonstrates a further difference between the factors designated CFII B and CFII B`. Whereas factor B required guanine nucleotide for activity, factor B` had substantial activity in the absence of nucleotide. The addition of Arf in the absence of GTPS produces no effect and a clear synergism between Arf and the two factors was observed in the presence of the activating nucleotide.


Figure 8: Differential requirement of GTPS by CFII B and CFII B` for activation of PLD activity. PLD activity was determined as described under ``Experimental Procedures.'' Assays contained 0.9 µg of PLD and as indicated, 10 µM GTPS, 0.5 µg of Arf, and various amounts of the cytosolic factors.



Evaluation of Potential CFII Factors

The ability of the non-Arf cytosolic factors to stimulate PLD activity in the presence of GTPS but the absence of Arf suggested the involvement of other GTP-binding proteins. At least one of these is the RhoA protein. When aliquots from the fractions eluted from hydroxylapatite (Fig. 4) were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies for RhoA (Fig. 9), the monomeric G protein was detected in fractions used for factor pools CFII A and CFII B. The other two pools, CFII B` and CFII C, were devoid of immunoreactivity for this protein. Actually, the presence or absence of RhoA was part of the basis for defining the two pools from column B. The fractions were also tested for the presence of Rho GDI (Fig. 9), a protein that inhibits GDP dissociation from Rho(26, 27) . Interestingly, this protein was only observed in the CFII B peak. The presence of Rho GDI in the B pool could explain its apparent lower activity relative to pool A in spite of a higher abundance of RhoA. Alternatively, pool A may contain another stimulatory component. The latter possibility is emphasized by the detection of RhoA in only the early fractions of the activity peak derived from high performance hydroxylapatite chromatography of pool A; stimulation by the latter fractions is indicative of a separate but, perhaps, similar factor.


Figure 9: Immunological detection of RhoA and Rho GDI in the CFII pools. Aliquots (5 µl) of indicated fractions from hydroxylapatite chromatography of pools A, B, and C shown in Fig. 4 were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis with the indicated antibodies as described under ``Experimental Procedures.'' The left lanes contained 60 ng of recombinant RhoA (rRhoA) or Rho GDI as indicated. Load refers to the pool from heparin-Sepharose, which was applied to the respective column being examined.



Fractions from the gel filtration procedure (Fig. 2) and the isolation on heparin-Sepharose (Fig. 3) were also blotted for the presence of RhoA (data not shown). As anticipated, the protein migrated with the higher mobility activity during gel filtration. Both peaks A and B from the heparin column also contained RhoA as anticipated. RhoA was not detected in the preparations of either Arf or the enriched PLD used for these experiments.

Effect of Recombinant RhoA, Cdc42, and Other Selected Monomeric G Proteins on PLD Activity

While RhoA was present in stimulatory fractions, the effect could be due to the presence of other GTP-binding proteins. Therefore, recombinant RhoA, produced in Sf9 cells, was tested for its ability to stimulate enriched PLD activity (Fig. 10). Activation of PLD activity was observed with concentrations of the protein in the low nanomolar range. Unfortunately, an unidentified inhibitory activity in the preparation prevented observation of the protein at higher concentrations, which might provide greater activation. Of equal interest was the ability of RhoA to show synergistic activation of PLD when combined with Arf in the form of myrArf5. The effects of RhoA, but not Arf, were attenuated by the addition of Rho GDI, presumably by inhibiting nucleotide exchange on RhoA. The relatively modest activity of RhoA alone and the enhancement of Arf activity resembles the action of CFII A and CFII B, the cytosolic factor pools that contain this small G protein. However, Rho GDI was only partially effective in blocking the action of CFII A (data not shown), consistent with the likelihood that there is another stimulatory factor in this pool.


Figure 10: Activation of PLD with recombinant RhoA and Cdc42. Enzyme activity was measured as described under ``Experimental Procedures,'' except that the reactions were performed in a 60-µl volume. Assays contained 1 µM GTPS, 2.5 µg of PLD, and the indicated concentrations of recombinant RhoA or Cdc42 either in the presence or absence of 35 nM myrArf5. As indicated, assays also contained 380 nM Rho GDI.



The recombinant RhoA from Sf9 cells is modified, at least in part, by isoprenylation of its C terminus(18) . In order to assess the requirement for this modification, a mutant RhoA, which contained the Val-14 activation mutation, was expressed in E. coli(18, 21) , purified, and mixed with PLD. Only very low activity (2-5 pmol of PC hydrolyzed) was observed, even at high concentrations (0.5-2 µM) of the nonmodified protein. A small synergism was observed when Arf was added in conjunction with increasing concentrations of the rRhoA(Val-14) (57-71 pmol of PC hydrolyzed versus 50 pmol with Arf alone). This indicates that the nonmodified protein had activity but that it was poor relative to the RhoA expressed in Sf9 cells, which is posttranslationally modified, minimally with an isoprenyl group.

While RhoA may account for at least some of the activity of CFII pools A and B, it does not explain the additional activity of the CFII A pool or the activities of the more robust CFII B` pool and that of the CFII C pool. Other selected monomeric G proteins have been tested for potential action on PLD. Cdc42, a member of the Rho family of monomeric G proteins, proved an effective activator of PLD activity and showed similar synergistic activity with added Arf (Fig. 10). The action of this protein could also be attenuated by Rho GDI. Other proteins that were tried but proved ineffective are shown in . Nonprenylated Rab1a, Rab3a, or Rab5 had essentially no activity by themselves or in the presence of native Arf or the CFII peak from gel filtration. The prenylated Rab1a was similarly unresponsive in comparison with RhoA. Interestingly, RhoA showed synergism with both Arf and the CFII fractions in which it resides, albeit at lower concentrations. The latter result resembles the interactive effects of the CFII pools (A and B) that contain RhoA with the pools (B` and C), which are devoid of this G protein (Fig. 6).


DISCUSSION

A phospholipase D activity has been described that can be activated with the monomeric G protein, Arf(9, 10) . The development of an in vitro assay, which uses exogenous substrate (9) to assay this activity, has allowed extensive purification of the enzyme as described in the companion report(13) . Use of enriched preparations of porcine PLD and reexamination of the stimulation provided by cytoplasmic Arf revealed the presence of other stimulatory factors in cytosol. Resolution of these activities yielded at least three unique components; Arf, RhoA, and a third factor that has not yet been identified. The identification of the Rho protein was anticipated by two previous reports that implicated Rho in the function of PLD. Thus, Bowman et al.(11) showed that the addition of Rho GDI inhibited a PLD activity observed in human neutrophils. More recently, addition of RhoA to plasma membrane preparations derived from rat liver caused a 2-fold enhancement of basal PLD activity(12) . The inability of the latter group to remove Arf or Cdc42 from their membrane preparation presumably precluded observation of stimulatory effects by these two proteins. The efficacy of recombinant RhoA, or the cytoplasmic factor pools that contained the native protein on the enriched PLD activity used in this study suggests the likelihood that this action of RhoA is a direct interaction with the PLD enzyme. The efficacy of Cdc42, a protein related to RhoA, suggests that stimulation of PLD activity may be a general functional property of the Rho family. Since the regulation by Arf suggests a potential role for PLD in protein traffic, other speculative candidates for regulation of this enzyme would include the Rab family of small G proteins. Exploration of various members of this and other monomeric G protein families has just begun.

The effect of RhoA by itself was relatively modest. Its efficacy is most apparent in its ability to facilitate activation of PLD by Arf. This effect could be achieved by enhancing activation of the Arf protein itself. However, significant stimulation induced by Rho alone indicates a more direct action on the PLD; it is more likely that Rho acts through PLD to increase interaction of the enzyme with Arf. One caveat to the action of RhoA is that a complete titration of the protein could not be achieved with current preparations of recombinant protein. It is plausible that higher concentrations of the monomeric G protein will yield more robust stimulation of PLD in the absence of Arf.

This pattern of regulation was repeated in the interaction between Arf and the cytosolic factor pools that did not contain RhoA. In this case, the CFII B` pool showed substantial activity on its own. It also had synergistic effects when combined with the factor pools containing RhoA. The identity of this third factor is unknown. Since a significant proportion of its activity could be obtained in the absence of guanine nucleotides, it is likely to represent more than another G protein, and this factor(s) is likely to facilitate PLD activity by another mechanism. One explanation for the synergism between Arf and CFII B` would be that the latter acts as a catalyst for nucleotide exchange on Arf. While this is possible, the effect of the factor on PLD activity in the absence of nucleotide suggests a more direct mechanism. A second possibility for synergism is that these factors would prevent degradation of the substrate by other contaminating activities such as a phospholipase A. This seems unlikely in view of the linearity of the assay (see Fig. 6in the companion paper(13) ), and no evidence of nonspecific substrate degradation or preservation was observed when PC labeled in the acyl moieties was utilized. Finally, the enhancement of stimulation by CFII B` by GTPS may be due to the coincident presence of a stimulating G protein in the CFII B` pool or to the potential presence of a GTP-binding protein associated with the PLD preparation itself but which is not effective in the absence of other factors.

The molecular mechanism behind the requirement for PIP(9, 13) in the expression of Arf-dependent PLD activity is unknown. The discernment of RhoA as a regulatory factor for this activity may offer part of the explanation. The dissociation of Rac from Rho GDI was stimulated by acidic phospholipids; PIP was particularly effective(28) . If this is also the case with RhoA, PIP could facilitate its activation in the CFII B preparation. Interestingly, acidic lipids, especially PIP, had marked effects on activation of Arf(29) . In contrast to these results, acidic lipids other than PIP and phosphatidylinositol 3,4,5-trisphosphate (PIP) are not effective in the PLD assay. It is possible, if not probable, that PIP has multiple effects; facilitation of G protein activation is one of these that contributes to the overall mechanism of regulation observed in these in vitro experiments.

What is the significance of the regulation of this enzyme by Arf, the Rho family, and potentially other GTP-dependent or independent proteins? The role of Arf in intracellular protein traffic suggests that PLD plays an important role in these processes, perhaps by modulating membrane lipids to facilitate formation or fusion of transport vesicles. RhoA and Cdc42 have been shown to mediate different cytoskeletal-associated assembly processes; thus, RhoA can regulate the formation of actin stress fibers and focal adhesions(21) , and Cdc42 is essential for bud site assembly in Saccharomyces cerevisiae (30, 31). RhoA has also been implicated in the regulation of phosphatidylinositol 3-kinase (32) and phosphatidylinositol 4-phosphate 5-kinase (18) activities. Evidence indicates that Cdc42 activates a phosphatidylinositol 3-kinase (33) as well as a serine/threonine-protein kinase(34) . It would seem more than coincidence that Rho and/or Cdc42 would regulate synthesis of PIP and PIP and that both these regulatory proteins and PIP/PIP facilitate the activity of PLD. This may indicate that this lipase is one of the key mediators of the action of the Rho family in cells.

Overall, the results delineated in this study indicate that PLD activity can be regulated by a variety of cytoplasmic factors. It is likely that membrane factors yet to be identified also impinge on this enzyme. These data then predict that activation of the enzyme and its sequelae mediate effects of multiple cellular pathways. It is possible that regulation of PLD by several factors is indicative of the use of this enzyme by a variety of signal transduction systems to achieve different end points in remote locations in the cell. Alternatively, the potential synergistic effects of these regulatory molecules suggest a dynamic capability of the enzyme to provide a robust response in the presence of multiple intracellular stimuli or the need for several interacting components to be stimulated before marked activity is initiated. This would allow the system to perform in a highly cooperative fashion. Such a response might be most desirable if this enzyme is involved in regulated secretory events as proposed by Cockcroft (35) or in controlled budding or fusion of vesicles during protein transport. The synergism between Arf and Rho raises the intriguing possibility that the functions of these proteins are interrelated in a fashion that remains to be discerned.

  
Table: Effect of monomeric G proteins on PLD activity



FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grants GM31954 (to P. C. S.) and GM44428 (to G. M. B.) and National Research Service Awards GM15359 (to W. D. S.) and GM15817 (to H. A. B.). Support was also obtained from 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.

The abbreviations used are: PLD, phospholipase D; GTPS, guanosine 5`-O-(3-thiotriphosphate); GDI, GDP dissociation inhibitor; CFII, cytoplasmic factor II, cytosolic activators of phospholipase D other than Arf; G protein, GTP-binding regulatory protein; myrArf5, myristoylated recombinant Arf5; TPCK, tosylphenylalanyl chloromethyl ketone; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; PIP, phosphatidylinositol 4,5-bisphosphate; PIP, phosphatidylinositol 3,4,5-trisphos-phate; PC, phosphatidylcholine.


ACKNOWLEDGEMENTS

We thank John Long and Steve Gutowski for excellent technical assistance and Kim Edwards for help in the preparation of this manuscript. We also thank Miguel Seabra for providing recombinant Rab proteins and Tsung Chuang for preparing recombinant RhoA and Rho GDI.

Addendum-During the review of this manuscript, a communication by Lambeth et al.(36) was published that provided evidence for an unidentified activity from cytosol, which could stimulate PLD activity in neutrophil membranes and potentiate the effects of Arf.


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