Mastoparan Selectively Activates Phospholipase D2 in Cell Membranes*

Ahmed ChahdiDagger §, Wahn Soo ChoiDagger ||, Young Mi KimDagger **, and Michael A. BeavenDagger DaggerDagger

From the Dagger  Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1760, the § Medical College of Wisconsin, Department of Medicine (Nephrology), Milwaukee, WI 53226, and the || Department of Immunology, College of Medicine, Konkuk University, Chungcheongbuk-Do 380-701, Korea

Received for publication, November 27, 2002, and in revised form, January 27, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Both known isoforms of phospholipase (PL) D, PLD1 and PLD2, require phosphatidylinositol 4,5-bisphosphate for activity. However, PLD2 is fully active in the presence of this phospholipid, whereas PLD1 activation is dependent on additional factors such as ADP-ribosylation factor-1 (ARF-1) and protein kinase Calpha . We find that mastoparan, an activator of Gi and mast cells, stimulates an intrinsic PLD activity, most likely PLD2, in fractions enriched in plasma membranes from rat basophilic leukemia 2H3 mast cells. Overexpression of PLD2, but not of PLD1, results in a large increase in the mastoparan-inducible PLD activity in membrane fractions, particularly those enriched in plasma membranes. As in previous studies, expressed PLD2 is localized primarily in the plasma membrane and PLD1 in granule membranes. Studies with pertussis toxin and other agents indicate that mastoparan stimulates PLD2 independently of Gi, ARF-1, protein kinase C, and calcium. Kinetic studies indicate that mastoparan interacts synergistically with phosphatidylinositol 4,5-bisphosphate and that oleate, itself a weak stimulant of PLD2 at low concentrations, is a competitive inhibitor of mastoparan stimulation of PLD2. Therefore, mastoparan may be useful for investigating the regulation of PLD2, particularly in view of the well studied molecular interactions of mastoparan with certain other strategic signaling proteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase D (PLD),1 which catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid, is present in most mammalian cells, where it is presumed to serve important roles in cell function (1-3). Two mammalian isoforms have been cloned, namely PLD1 (as two variants, PLD1a and PLD1b) and PLD2, both of which require phosphatidylinositol (PI) 4,5-bisphosphate for activity (4-7). In the presence of PI 4,5-bisphosphate, PLD1 can be activated by several mechanisms. These include activation by small GTPases such as ARF and Rho proteins (8-12), Rho kinase (13), calcium/calmodulin-dependent protein kinase II (14), and protein kinase C (PKC) in a catalytically dependent and independent manner (8, 15, 16).

In contrast to PLD1, PLD2 is activated by PI 4,5-bisphosphate alone, and this activation is not affected by the small GTPases or PKCalpha (4) and only weakly by ARF proteins (17, 18). PLD2 activity could conceivably be regulated in vivo by PLD2 inhibitory proteins such as the synucleins (4, 19) or by recruitment of PI 4-phosphate 5-kinase 1alpha , the enzyme responsible for the intracellular synthesis of PI 4,5-bisphosphate. It has been shown that intracellular production of PI 4,5-bisphosphate is coupled to PLD activation (20-23) and that PI 4-phosphate 5-kinase 1alpha and PLD2 interact and co-localize when these two molecules are co-expressed in cells (24). However, there is no unambiguous evidence that any of these mechanisms operate in physiologically stimulated cells. Oleate at sub-millimolar concentrations stimulates PLD2 activity in the absence of PI 4,5-bisphosphate and synergistically stimulates PLD2 activity in the presence of PI 4,5-bisphosphate (25, 26). At high millimolar concentrations, oleate inhibits PLD2 as well as PLD1 activities (17, 27). The physiological significance of the effects of oleate on PLD2 is unclear.

PLD appears to be essential for stimulated secretion of granules from mast cells. Studies with pharmacologic agents in the rat (RBL-2H3) mast cell line show that PLD activation correlates closely with secretion under a variety of circumstances (28, 29) and that primary alcohols, which divert the production of phosphatidic acid by PLD to phosphatidylalcohol (referred to as a transphosphatidylation reaction), suppress secretion as well (20, 28). Moreover, the secretory response to antigen can be reconstituted in permeabilized RBL-2H3 cells by provision of ARF-1 or the phosphatidylinositol transfer protein, either of which increases levels of phosphatidylinositol 4,5-bisphosphate and thereby restores PLD activity (20). Both isoforms of PLD are present in RBL-2H3 cells (30). Expression studies with wild type and mutant forms of the PLDs suggest that both isoforms participate in the secretory process (30) and that PLD1 is expressed primarily on the granule membrane and PLD2 on the plasma membrane (30, 31).

Mastoparan, a cationic tetradecapeptide and a mast cell stimulant, can assume an amphipathic alpha -helical conformation in a hydrophobic environment and is known to mimic the interactions of physiologic proteins with target proteins such as trimeric G proteins (32) and calmodulin (33, 34). It has also been reported that mastoparan inhibits ARF1-stimulated PLD activity (35), now recognized as PLD1, in cytosol-depleted cells, although more recent reports suggest that mastoparan is a stimulant of PLD in intact cells (36-38). While investigating the mechanisms of action of mastoparan and other compounds thought to stimulate mast cell secretion through the G protein Gi (39), we found that mastoparan is a uniquely strong stimulant of PLD in the cultured RBL-2H3 mast cells. These and additional studies with membrane fractions from normal RBL-2H3 cells and cells made to overexpress PLD1 or PLD2 show that mastoparan selectively activates PLD2 in the plasma membrane independently of any action on Gi.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The following materials were purchased from the indicated sources. Mastoparan and other secretagogues came from Bachem (Torrance, CA), L-alpha -phosphatidyl-D-myo-inositol 4,5-bisphosphate (PI 4,5-bisphosphate) from Roche Molecular Biochemicals, GDPbeta S, GTPgamma S, L-alpha -dipalmitoylphosphatidylcholine, and oleic acid from Sigma, phosphatidylethanolamine from Avanti Polar Lipids (Alabaster, Al), [choline-methyl-3H]dipalmitoylphosphatidylcholine (50 µCi/mmol) from PerkinElmer Life Sciences, pertussis toxin from List Biologicals (Campbell, CA), cell culture reagents and 60% Iodixanol (OptiPrep) from Invitrogen, and Tris-glycine polyacrylamide gels from Novex (San Diego, CA).

Cell Culture and Experimental Conditions-- RBL-2H3 cells were grown as monolayers in minimal essential medium with Earle's salts and supplemented with glutamine, antibiotics, and 15% fetal bovine serum (40). Where indicated, pertussis toxin (0.2 µg/ml) was added 3 h before the experiment or the lysis of cells for the preparation of membrane fractions.

Measurement of PLD Activity in Intact Cells by the Transphosphatidylation Assay-- RBL-2H3 m1 cells were incubated in complete growth medium in 24-well plates, and [3H]myristic acid, 2 µCi/ml, was added for the final 90 min of incubation. Cells were then incubated in PIPES-buffered medium (30) in the presence of 1% ethanol for 10 min before stimulation. Under these conditions, metabolically stable [3H]phosphatidylethanol is produced at the expense of [3H]phosphatidic acid by transphosphatidylation, a catalytic reaction that is unique to PLD (41). Radiolabeled phosphatidylethanol was isolated and quantified by minor modifications of previously described procedures (42). The reaction was terminated by the addition (0.75 ml/well) of a mixture of chloroform/methanol/4 N HCl (100:200:2) (v/v/v) to form a single phase that was subsequently separated into two phases by the addition of 0.25 ml of chloroform, which contained 30 µg each of unlabeled phosphatidic acid and phosphatidylethanol as well as 0.25 ml of 0.1 N HCl. A 0.5-ml sample of the lower chloroform phase was evaporated to dryness under nitrogen. The residue was dissolved in 100 µl of a mixture of chloroform/methanol (2:1) of which 25 µl was placed on a silica gel-plated sheet for thin layer chromatography in a mixture of chloroform/methanol/glacial acetic acid (65:15:2) (v/v/v) (43). The sheet was air-dried and then exposed to iodine vapor to visualize phospholipids. Regions containing phosphatidic acid and phosphatidylethanol were excised for an assay of tritium.

Transient Transfection of Cells with HA-tagged PLDs-- HA-tagged plasmids for PLD were kindly supplied by Dr. Michael A. Frohman (Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York, 11794-8651). For confocal microscopy (30), full-length cDNA were excised by SmaI and HpaI for PLD1b and SmaI and XbaI for PLD2 and subcloned into a pEGFP-C expression vector, Clontech. Cells were transiently transfected with each DNA preparation (25 µg/2 × 107 cells) by electroporation (Bio-Rad Gene PulserTM, 960 µF, 250 V). Approximately 25% of cells were transfected by this procedure. Successful transfection was confirmed by Western blotting. Cells were used within 48 h of transfection.

Subcellular Fractionation-- Cultured RBL-2H3 cells (107 cells) were detached from confluent cultures with 20 mM EDTA in ice-cold phosphate-buffered saline. Cells were pelleted (1,000 × g for 10 min) and resuspended in 2 ml of homogenization buffer (10 mM HEPES, pH7.4, 1 mM EDTA, 0.25 M sucrose, supplemented with a protease inhibitor mixture). The cells were disrupted in a Dounce homogenizer (15 strokes). Nuclei and unbroken cells were removed by centrifuging the homogenate at 3,000 × g for 10 min, resuspending the pellet in 2 ml of buffer, and repeating the centrifugation step. The supernatant (postnuclear) fractions from both centrifugation steps were combined and centrifuged at 80,000 × g for 1 h. The postnuclear pellet was resuspended in 1.0 ml of homogenization buffer. All operations were carried out at 4 °C.

A stock solution of 50% iodixanol was prepared by diluting OptiPrep with a solution of 0.25 M sucrose, 6 mM EDTA, and 60 mM HEPES (pH7.4) from which linear gradients of 1-20% iodixanol were formed using a gradient maker exactly as described (44). The resuspended postnuclear fraction was loaded on top of the gradient and centrifuged in a Beckman SW41 rotor at 200,000 × g for 3 h at 4 °C. Sequential 1-ml fractions (gradient fractions) were then collected from the bottom of the gradient for an assay of subcellular markers and HA-tagged PLD as described below.

Ten microliters from each gradient fraction were mixed with Laemmli buffer, and the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blotted for calnexin (anti-calnexin C terminus polyclonal antibody, Stress Gen, Victoria, British Columbia, Canada), rat mast cell protease II (anti-RMCP II antibody from Moredun Animal Health, Midlothian, Scotland), and Fcepsilon RIbeta (antibody was a gift from Dr. Juan Rivera, NIAMS, National Institutes of Health) for the detection of endoplasmic reticulum, granule membrane, and plasma membrane, respectively. The relative amounts of protein were assessed by densitometric scanning. A procedure based on the addition of [3H]galactose onto the oligosaccharide moieties of ovomucoid was used for the assay of galactosyltransferase activity in Golgi-containing fractions as described by others (45).

Assay of PLD in Subcellular Fractions-- Postnuclear and gradient fractions were assayed for HA-tagged PLDs by gel electrophoresis and immunoblotting (anti-HA antibody from Upstate Biotechnology, Lake Placid, NY) as described above and for PLD activity. PLD activity was assayed by the measurement of [3H]choline release from the PLD substrate [choline-methyl-3H]dipalmitoylphosphatidylcholine exactly as described by Massenburg et al. (46). For this assay, 20 µl of each fraction (or the amount indicated) was added to 25 µl of a vesicle preparation of mixed phospholipids that contained 140,000 dpm of [choline-methyl-3H]dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, PI 4,5-bisphosphate, and phosphatidylethanolamine and a buffer solution to make a final volume of 125 µl. Mastoparan or other reagents were added where noted in the text or figure legends. For one set of experiments (Fig. 6), the amounts of dipalmitoylphosphatidylcholine or PI 4,5-bisphosphate were altered to give the designated concentration of lipid. The mixture was incubated at 37 °C for 1 h before the addition of 1 ml of chloroform/methanol/concentrated HCl (50:50:0.3) (v/v/v), and 0.35 ml of 1 M HCl/5 mM EGTA. The upper aqueous phase was assayed for [3H]choline by liquid scintillation counting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mastoparan Stimulates PLD in Cell Membrane Fractions-- An assay of PLD activity in intact RBL-2H3 cells by the transphosphatidylation assay (see "Experimental Procedures") showed that mastoparan was a particularly strong stimulant of PLD when compared with its inactive analog, mastoparan 17, and other agents thought to stimulate mast cell degranulation via Gi (Fig. 1A). The assay of PLD activity in vitro by the [3H]choline release assay showed that the mastoparan-stimulated PLD activity appeared to reside in the membrane pellet fraction of these cells (Fig. 1B). Separation of the postnuclear membrane fraction into different fractions by density gradient centrifugation revealed that the mastoparan-stimulated PLD activity varied among these fractions (Fig. 2A). The mastoparan-stimulated activity appeared to be distributed in fractions enriched with the plasma membrane marker (Fcepsilon RIbeta ) and, perhaps to a lesser extent, the granule marker (RMCPII) (Fig. 2B). In the same experiments, ARF-1/GTPgamma S elicited a modest stimulation of a PLD activity, presumed to be PLD1, primarily in fractions enriched in endoplasmic reticulum (calnexin; fractions 1 and 2) and granule membranes (RMCPII; fractions 9-12). The extent of stimulation was at least ten times less than that achieved with mastoparan (Fig. 2A). These studies indicated that mastoparan was a remarkably strong stimulant of PLD activity in vivo and in vitro and that the stimulated PLD activity was unlikely to be PLD1.


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Fig. 1.   Mastoparan, and not other mast cell secretagogues, is a robust stimulant of PLD activity in intact RBL-2H3 cells and membrane preparations from these cells. A, RBL-2H3 cells were incubated with [3H]myristic acid in growth medium for measurement of PLD activity by the transphosphatidylation assay as described under "Experimental Procedures." The medium was replaced with a PIPES-buffered medium containing 1% ethanol. Cells were then exposed to vehicle (NS), 30 µM mastoparan (MP), 30 µM mastoparan-17 (MP-17, an inactive analog of mastoparan), 100 µM substance P (SP), 1 µM neuropeptide Y (C-terminal amino acids 18-36) (NPY), or 10 µg/ml compound 48/80 for 10 min at 37 °C for measurement of the PLD product, [3H]phosphatidylethanol. B, cytosolic and post-nuclear membrane pellet fractions from RBL-2H3 cells were assayed for PLD activity in vitro by the [3H]choline release assay as under "Experimental Procedures" in the absence or presence of 30 µM mastoparan. The data are mean ± S.E. of values from three experiments.


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Fig. 2.   Distribution of mastoparan-stimulated PLD activity in cell fractions. The post-nuclear pellet fraction from RBL-2H3 cells was fractionated further by density gradient centrifugation. The twelve fractions thus obtained were assayed for PLD activity by the [3H]choline release assay in the absence or presence of ARF-1 (1.5 µM) plus GTPgamma S (30 µM) or mastoparan (30 µM) (panel A) and for cell markers (panel B). The markers were calnexin (for the endoplasmic reticulum, ER), Fcepsilon RIbeta chain (for the plasma membrane, PM), galactosyltransferase activity (for the Golgi), and rat mast cell protease II (for granules). The data show the proportion (as percent) of each marker in the individual fractions. The experiment shown was typical of three such experiments.

Mastoparan Activates Expressed HA-tagged PLD2, but Not PLD1, in Post-nuclear Membrane Fractions-- Transient expression studies showed that mastoparan had little or no stimulatory activity on supernatant or membrane fractions from cells containing gene vector or cells made to express HA-PLD1 when compared with cells made to express HA-PLD2 (Fig. 3A). In the latter cells, the membrane fraction, but not the supernatant fraction, exhibited an extraordinarily high PLD activity in response to mastoparan (Fig. 3A). This response was ~15-fold greater than that achieved with ARF-1/GTPgamma S in the same membrane fraction. The selective activation of PLD2 was also apparent in studies with COS7 cells made to express HA-PLD1 or HA-PLD2. As in RBL-2H3 cells, mastoparan produced a robust stimulation of PLD only in the membrane fraction from COS7 cells that expressed HA-PLD2 (Fig 3B).


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Fig. 3.   Mastoparan stimulates expressed HA-PLD2 in cell membranes independently of Gi, PKC, and calcium. RBL-2H3 cells were transiently transfected with genes for HA-vector, HA-PLD1, or HA-PLD2. PLD activity was assayed by the measurement of [3H]choline release in the supernatant (SN) and post-nuclear membrane (P) fractions in the presence of mastoparan (30 µM) or ARF-1 (1.5 µM) plus GTPgamma S (30 µM) (panel A). For all remaining panels, PLD assays were performed in the presence of mastoparan (30 µM). For panel B, assays of PLD activity were performed on the supernatant and post-nuclear membrane fractions from COS7 cells transiently transfected with vector, HA-PLD1, and HA-PLD2. For panel C, the membrane fractions were prepared from cells treated with 0.2 µg/ml pertussis toxin (PTx) for 3 h or left untreated (Control) before lysis. For panel D, the membrane fractions were prepared from RBL-2H3 cells made to express HA-PLD2 and assayed for PLD activity in the absence or presence of 10 µM Ro 31-7549 (Ro-31) or 1 mM EGTA and from cells made to express HA-PLD2 and treated with pertussis toxin. Data are the mean ± S.E. of values from three experiments.

Prior treatment of cells with pertussis toxin to inactivate the alpha -subunit of Gi, a primary target of mastoparan in mast cells (39), failed to diminish the PLD response to mastoparan in intact cells (data not shown) or in membrane fractions collected from cells made to express HA-PLD2 (Fig. 3C). Mastoparan stimulation of HA-PLD2 in membrane fractions was also undiminished in the presence of the PKC inhibitor Ro 31-7549 (47, 48), and EGTA (Fig. 3D). Collectively, the results indicated that mastoparan stimulation of membrane PLD2 in vitro is independent of Gi, PKC, and calcium. These results were consistent with previous reports that mastoparan stimulation of PLD in intact cells was unaffected by inhibitors of G-proteins (37) and PKC or by chelation of extracellular calcium (36).

Mastoparan-sensitive PLD2 Is Located in the Plasma Membranes-- In additional experiments with cells made to express HA-PLD2, separation of membrane fractions by density gradient demonstrated that the distribution of HA-PLD2 (Fig. 4A) and mastoparan-stimulated PLD2 activity (Fig. 4B) overlapped with the distribution of the endoplasmic reticulum marker (calnexin) and plasma membrane marker (Fcepsilon RI). Although the fit was not perfect, the distribution of HA-PLD2 and mastoparan-stimulated activity appeared to best correlate with the distribution of Fcepsilon RIbeta with maximum amounts of each in fraction 5. Consistent with previous studies (26, 30), however, fluorescence microscopy of cells made to express PLDs tagged with green fluorescence protein showed that PLD2 was located largely in the plasma membrane, whereas PLD1 was located largely on granules (Fig. 4C).


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Fig. 4.   Expressed PLD2 is located predominantly in the plasma membrane. Post-nuclear cell membrane fractions from HA-PLD2 transfected cells were fractionated by density gradient centrifugation as described for Fig. 1 and blotted for HA-PLD2 and cell markers (panel A). Peak fractions were also assayed for PLD activity by measurement of [3H]choline release (Panel B). In concurrent studies, PLD1 and PLD2 tagged with green fluorescent protein were expressed to confirm the cellular locations of the PLDs by confocal microscopy (Panel C).

Enzymatic Characteristics of PLD2 Activation by Mastoparan and the Interactions with PI 4,5-Bisphosphate-- Additional features of the PLD2 response to mastoparan are shown in Fig. 5. In the assay system used, near maximal release of [3H]choline from [choline-methyl-3H]dipalmitoylphosphatidylcholine was observed with as little as 0.1 µg of membrane protein obtained from cells made to express HA-PLD2, whereas comparatively little release was observed with up to 3 µg of membrane protein in membrane fractions obtained from cells made to express HA-PLD1 or the HA vector (Fig. 5A). Significant and maximal release occurred with 1 and 30 µM mastoparan, respectively (Fig. 5B). With 30 µM mastoparan, release was initially rapid but continued for up to 80 min (Fig. 5C). Approximately 30% of the substrate was consumed during the course of these experiments. On the assumption that this 30% was the maximum amount of substrate that was available for hydrolysis by PLD2, the analysis of the data in Fig. 5C indicated second order reaction kinetics with half-lives of 2 and 45 min for each rate component. Regardless of the reason for the second order component, we presume that any inactivation of PLD2 during the course of incubation must be as slow or slower than the second component.


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Fig. 5.   Characteristics of mastoparan stimulation of expressed HA-PLD2 in membrane fraction. The membrane fraction from RBL-2H3 cells made to express vector, HA-PLD1, or HA-PLD2 were assayed for PLD activity in the presence of 30 µM mastoparan (Panels A and C) or at the concentration indicated (Panel B). Assays were performed with 1 µg of membrane protein except in panel A, where the amount of protein was varied. Data are the mean ± S.E. of values from three experiments.

Additional studies with the membrane fraction from HA-PLD2-transfected cells suggested that mastoparan did not alter the affinity or availability of substrate for PLD2 but rather the rate of release of [3H]choline in response to the addition of PI 4,5-bisphosphate. Measurement of the rate of release with different concentrations of substrate (Fig. 6A) and replotting the data as a double-reciprocal plot (inset, Fig. 6A) indicated that the maximal rate (Vmax) was increased, whereas the affinity (apparent Km) was unchanged. When the concentration of PI 4,5-bisphosphate was altered (Fig. 6B, open symbols), near maximal rates of [3H]choline release were observed with 6 µM PI 4,5-bisphosphate (the concentration used in previous assays). In the presence of mastoparan, release of [3H]choline was enhanced ~3.5-fold (Fig. 6B, solid symbols). Mastoparan by itself (i.e. 30 µM mastoparan at 0 µM PI 4,5-bisphosphate in Fig. 6B) stimulated membrane PLD2 to the same extent as 6 µM PI 4,5-bisphosphate and thus acted in synergy together with PI 4,5-bisphosphate. The shapes of the two curves in Fig. 6B suggested that the affinity (i.e. half Vmax) of PI 4,5-bisphosphate for PLD was unaltered.


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Fig. 6.   Effects of mastoparan on the dependence of PLD2 activity on substrate and PI 4,5-bisphosphate. The rates of [3H]choline release were determined with membrane fractions (1 µg of protein) from RBL-2H3 cells made to overexpress HA-PLD2 at various concentrations of dipalmitoylphosphatidylcholine (DDPC) (panel A) or PI 4,5-bisphosphate (PIP2) (panel B). The concentration of PI 4,5-bisphosphate was 6 µM in panel A, and the concentration of dipalmitoylphosphatidylcholine was 4 µM in panel B. Measurements were performed in the presence or absence of 30 µM mastoparan as indicated. The inset in panel A depicts a double-reciprocal plot of the data to show that regression lines intersect at the abscissa; only three data points are shown for clarity, but the regression lines were calculated from all data points. Data are the mean ± S.E. of values from three experiments.

Mastoparan Activation of PLD2 Is Inhibited by Oleate-- In view of the reported synergy between oleate and PI 4,5-bisphosphate in the activation of PLD2 (25), we tested the effects of oleate on mastoparan stimulation of PLD2 in the membrane fraction from HA-PLD2-transfected cells. Oleate in the presence of PI 4,5-bisphosphate caused some additional stimulation of PLD2 at 0.1 mM and inhibited PLD2 activity at higher concentrations consistent with previous reports (inset, Fig. 7A). Mastoparan was a much more robust stimulant than oleate. However, the stimulation of PLD2 by mastoparan was inhibited by oleate in a dose-dependent manner (Fig. 7). The parallel shift in curves (as in Fig. 7) could be indicative of competitive inhibition by oleate.


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Fig. 7.   Oleate inhibits mastoparan stimulation of PLD2. The rates of [3H]choline release were determined with membrane fractions (1 µg of protein) from RBL-2H3 cells made to overexpress HA-PLD2 at various concentrations of mastoparan in the absence or presence of the indicated concentrations of oleate. Standard concentrations of dipalmitoylphosphatidylcholine (4 µM) and PI 4,5-bisphosphate (6 µM) were used for these determinations. The inset shows [3H]choline release in the presence of oleate alone, and the values for this release have been subtracted from the values plotted in the main panel. Data are the mean ± S.E. of values from three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mastoparan activates endogenous PLD in intact RBL-2H3 cells and in isolated preparations enriched in plasma membranes where PLD2 is located. This activation was apparent when PLD activity was assessed by the transphosphatidylation assay in intact cells and by a [3H]choline release assay in membrane preparations. The studies with expressed PLD isoforms confirm that mastoparan selectively activates PLD2 in membrane preparations in both RBL-2H3 and COS7 cells. The activation of PLD2 is not dependent on Galpha i, PKC, or calcium. This is in contrast to the stimulation of PLD and the secretion by compound 48/80 and other polybasic secretagogues in intact mast cells, where these responses are mediated via Galpha i, and are dependent on PKC or calcium (49, 50). Therefore, mastoparan has additional activities distinct from its ability to activate Galpha i; one of them apparently is the ability to activate PLD2 but not PLD1 in membrane preparations.

Mastoparan can assume an amphipathic alpha -helical conformation on passing from an aqueous to lipid environment and, by so doing, can mimic the interactions of physiologic alpha -helical proteins with key signaling molecules such as Gi with receptors (32) and calmodulin with calmodulin-binding proteins. With respect to calmodulin, mastoparan binds with exceptionally high affinity in a calcium-dependent manner (33) resulting in possible displacement of calmodulin-binding proteins (34). Mastoparan assumes a helical conformation within the globular Ca2+/calmodulin/mastoparan complex in a manner that is thought to reproduce the interaction of calmodulin with target proteins (51). Calmodulin has been proposed as a negative regulator of basal PLD2 activity on the basis of the effects of calmodulin inhibitors (52). Whether mastoparan unmasks latent PLD2 activity by disabling the inhibitory activity of calmodulin or that of other, better characterized inhibitors of PLD such as the synucleins (7) is unknown but worthy of further investigation. In particular, the synucleins possess conserved amphipathic alpha -helical domains (53), are potent inhibitors of PLD2 (4, 19), and are present in abundant amounts in RBL-2H3 membranes.2

It is possible also that mastoparan interacts directly with PLD2 because it can stimulate PLD2 in the absence of, and to the same extent as, PI 4,5-bisphosphosphate and yet act in synergy with PI 4,5-bisphosphate. Moreover, the studies with oleate and mastoparan (Fig. 7) suggest that these two agents compete for the same site and that occupation of this site by mastoparan and, to a much lesser extent, oleate, causes activation of PLD2 in the absence of PI 4,5-bisphosphate. The ability of mastoparan (this paper) and oleate (25) to act in synergy with PI 4,5-bisphosphate suggests further that the mastoparan/oleate-binding site is distinct from the PI 4,5-bisphosphate-binding site. In this context, oleate appears to act as a partial agonist or antagonist and mastoparan as a full agonist, because oleate inhibits stimulation of PLD2 by mastoparan. If this scenario is correct and the physiologic ligand for the mastoparan/oleate-binding site is a regulator of PLD2 activity, mastoparan might be a useful probe for studies of PLD2 in view of the well studied molecular interactions of mastoparan with other strategic signaling molecules.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These two authors contributed equally to this manuscript.

** Present Address: Dept. of Pharmacy, Duksung Women's University, 419 Ssangmoon-dong, Tobong-gu, Seoul 132-714, Korea.

Dagger Dagger To whom correspondence should be addressed: Rm. 8N109/ Bldg. 10, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1760. Tel.: 301-496-6188; Fax: 301-402-0171; E-mail: beavenm@nhlbi.nih.gov.

Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212084200

2 W. S. Choi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; ARF, ADP-ribosylation factor; Fcepsilon RI, high affinity receptor for the immunoglobulin IgE; HA, hemagglutinin A; PI, phosphatidylinositol; PIPES, 1,4-piperazinediethanesulfonic acid; PKC, protein kinase C; RBL, rat basophilic leukemia; RMCP II, rat mast cell protease II.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Exton, J. H. (1997) Physiol. Rev. 77, 303-320[Abstract/Free Full Text]
2. Liscovitch, M., Czarny, M., Fiucci, G., and Tang, X. (2000) Biochem. J. 345, 401-415[CrossRef][Medline] [Order article via Infotrieve]
3. Jones, D., Morgan, C., and Cockcroft, S. (1999) Biochim. Biophys. Acta 1439, 229-244[Medline] [Order article via Infotrieve]
4. Colley, W. C., Sung, T. C., Roll, R., Jenco, J., Hammond, S. M., Altshuller, Y., Bar-Sagi, D., Morris, A. J., and Frohman, M. A. (1997) Curr. Biol. 7, 191-201[Medline] [Order article via Infotrieve]
5. Steed, P. M., Clark, K. L., Boyar, W. C., and Lasala, D. J. (1998) FASEB J. 12, 1309-1317[Abstract/Free Full Text]
6. Millar, C. A., Jess, T. J., Saqib, K. M., Wakelam, M. J. O., and Gould, G. W. (1999) Biochem. Biophys. Res. Comm. 254, 734-738[CrossRef][Medline] [Order article via Infotrieve]
7. Frohman, M., Sung, T.-C., and Morris, A. (1999) Biochim. Biophys. Acta 1439, 175-186[Medline] [Order article via Infotrieve]
8. Hammond, S. M., Jenco, J. M., Nakashima, S., Cadwallader, K., Gu, Q. M., Cook, S., Nozawa, Y., Prestwich, G. D., Frohman, M. A., and Morris, A. J. (1997) J. Biol. Chem. 272, 3860-3868[Abstract/Free Full Text]
9. Park, S. K., Provost, J. J., Bae, C. D., Ho, W. T., and Exton, J. H. (1997) J. Biol. Chem. 272, 29263-29271[Abstract/Free Full Text]
10. Sung, T. C., Roper, R. L., Zhang, Y., Rudge, S. A., Temel, R., Hammond, S. M., Morris, A. J., Moss, B., Engebrecht, J., and Frohman, M. A. (1997) EMBO J. 16, 4519-4530[Abstract/Free Full Text]
11. Min, D. S., Park, S. K., and Exton, J. H. (1998) J. Biol. Chem. 273, 7044-7051[Abstract/Free Full Text]
12. Bae, C. D., Min, D. S., Fleming, I. N., and Exton, J. H. (1998) J. Biol. Chem. 273, 11596-11604[Abstract/Free Full Text]
13. Schmidt, M., Vob, M., Oude Weernink, P. A., Wetzel, J., Amano, M., Kaibuchi, K., and Jakobs, K. H. (1999) J. Biol. Chem. 274, 14648-14654[Abstract/Free Full Text]
14. Min, D. S., Cho, N. J., Yoon, S. H., Lee, Y. H., Hahn, S. J., Lee, K. H., Kim, M. S., and Jo, Y. H. (2000) J. Neurochem. 75, 274-281[CrossRef][Medline] [Order article via Infotrieve]
15. Zhang, Y., Altshuller, Y. M., Hammond, S. M., and Frohman, M. A. (1999) EMBO J. 18, 6339-6348[Abstract/Free Full Text]
16. Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., and Ryu, S. H. (1999) Biochemistry 38, 10344-10351[CrossRef][Medline] [Order article via Infotrieve]
17. Lopez, I., Arnold, R. S., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 12846-12852[Abstract/Free Full Text]
18. Sung, T. C., Altshuller, Y. M., Morris, A. J., and Frohman, M. A. (1999) J. Biol. Chem. 274, 494-502[Abstract/Free Full Text]
19. Jenco, J. M., Rawlingson, A., Daniels, B., and Morris, A. J. (1998) Biochemistry 37, 4901-4909[CrossRef][Medline] [Order article via Infotrieve]
20. Way, G., O'Luanaigh, N., and Cockcroft, S. (2000) Biochem. J. 346, 63-70[CrossRef][Medline] [Order article via Infotrieve]
21. Arneson, L. S., Kunz, J., Anderson, R. A., and Traub, L. M. (1999) J. Biol. Chem. 274, 17794-17805[Abstract/Free Full Text]
22. Siddhanta, A., Backer, J. M., and Shields, D. (2000) J. Biol. Chem. 275, 1023-12031[Abstract/Free Full Text]
23. Liscovitch, M., and Cantley, L. C. (1995) Cell 81, 659-662[Medline] [Order article via Infotrieve]
24. Divecha, N., Roefs, M., Halstead, J. R., D'Andrea, S., Fernandez-Borga, M., Oomen, L., Saqib, K. M., Wakelem, M. J. O., and D'Santos, C. (2000) EMBO J. 19, 5440-5449[Abstract/Free Full Text]
25. Kim, J. H., Kim, Y., Lee, S. D., Lopez, I., Arnold, R. S., Lambeth, J. D., Suh, P. G., and Ryu, S. H. (1999) FEBS Lett. 454, 42-46[CrossRef][Medline] [Order article via Infotrieve]
26. Sarri, E., Pardo, R., Fensome-Green, A., and Cockcroft, S. (2002) Biochem. J. 369, 319-329[CrossRef]
27. Kodaki, T., and Yamashita, S. (1997) J. Biol. Chem. 272, 11408-11413[Abstract/Free Full Text]
28. Cissel, D. S., Fraundorfer, P. F., and Beaven, M. A. (1998) J. Pharmacol. Exp. Ther. 285, 110-118[Abstract/Free Full Text]
29. Chahdi, A., Choi, W. S., Kim, Y. M., Fraundorfer, P. F., and Beaven, M. A. (2002) Mol. Immunol. 38, 1269-1276[CrossRef][Medline] [Order article via Infotrieve]
30. Choi, W. S., Kim, Y. M., Combs, C., Frohman, M. A., and Beaven, M. A. (2002) J. Immunol. 168, 5682-5689[Abstract/Free Full Text]
31. Brown, F. D., Thompson, N., Saqid, K. M., Clark, J. M., Powner, D., Thompson, N. T., Solari, R., and Wakelam, M. J. O. (1998) Curr. Biol. 8, 835-838[Medline] [Order article via Infotrieve]
32. Sukumar, M., and Higashijima, T. (1992) J. Biol. Chem. 267, 21421-21424[Abstract/Free Full Text]
33. Murase, T., and Iio, T. (2002) Biochemistry 41, 1618-1629[CrossRef][Medline] [Order article via Infotrieve]
34. Malencik, D., and Anderson, S. (1984) Biochemistry. 23, 2420-2428[Medline] [Order article via Infotrieve]
35. Fensome, A., Cunningham, E., Troung, O., and Cockcroft, S. (1994) FEBS Lett. 349, 34-38[CrossRef][Medline] [Order article via Infotrieve]
36. Mizuno, K., Nakahata, N., and Ohizumi, Y. (1995) Br. J. Pharmacol. 116, 2090-2096[Abstract]
37. Lee, S. Y., Park, N. G., and Choi, M. U. (1998) FEBS Lett. 432, 50-54[CrossRef][Medline] [Order article via Infotrieve]
38. Farquhar, M., Soomets, U., Bates, R. L., Martin, A., Langel, U., and Howl, J. (2002) Chem. Biol. 9, 63-70[CrossRef][Medline] [Order article via Infotrieve]
39. Mousli, M., Bueb, J. L., Bronner, C., Rouot, B., and Landry, Y. (1990) Trends Pharmacol. Sci. 11, 358-362[CrossRef][Medline] [Order article via Infotrieve]
40. Ali, H., Cunha-Melo, J. R., Saul, W. F., and Beaven, M. A. (1990) J. Biol. Chem. 265, 745-753[Abstract/Free Full Text]
41. Dennis, E. A., Rhee, S. G., Billah, M. M., and Hannun, Y. A. (1991) FASEB J. 5, 2068-2077[Abstract/Free Full Text]
42. Ali, H., Choi, O. H., Fraundorfer, P. F., Yamada, K., Gonzaga, H. M. S., and Beaven, M. A. (1996) J. Pharm. Exp. Ther. 276, 837-845[Abstract]
43. Tomhave, E. D., Richardson, R. M., Didsbury, J. R., Menard, L., Snyderman, R., and Ali, H. (1994) J. Immunol. 153, 3267-3275[Abstract/Free Full Text]
44. Zhang, J., Kang, D. E., Xia, W., Okochi, M., Mori, H., Selkoe, D. J., and Koo, E. H. (1998) J. Biol. Chem. 273, 12436-12442[Abstract/Free Full Text]
45. Bretz, R., and Staubli, W. (1977) Eur. J. Biochem. 77, 181-192[Medline] [Order article via Infotrieve]
46. Massenburg, D., Han, J. S., Liyanage, M., Patton, W. A., Rhee, S. G., Moss, J., and Vaughan, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11718-11722[Abstract/Free Full Text]
47. Wilkinson, S. E., Parker, P. J., and Nixon, J. S. (1993) Biochem. J. 294, 335-337[Medline] [Order article via Infotrieve]
48. Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756[Abstract/Free Full Text]
49. Chahdi, A., Fraundorfer, P. F., and Beaven, M. A. (2000) J. Pharmacol. Exp. Ther. 292, 122-130[Abstract/Free Full Text]
50. Senyshyn, J., Baumgartner, R. A., and Beaven, M. A. (1998) J. Immunol. 160, 5136-5144[Abstract/Free Full Text]
51. Brown, S., Martin, S., and Bayley, P. (1997) J. Biol. Chem. 272, 3389-3397[Abstract/Free Full Text]
52. Boyano-Adanez, M.C., and Gustavsson, L. (2002) Neurochem. Int. 40, 261-268[CrossRef][Medline] [Order article via Infotrieve]


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