From the 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
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ABSTRACT |
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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 C 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 PKC 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 Materials--
The following materials were purchased from the
indicated sources. Mastoparan and other secretagogues came from Bachem
(Torrance, CA),
L- 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 Fc 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.
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 (Fc 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/GTP
Prior treatment of cells with pertussis toxin to inactivate
the 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 (Fc 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.
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.
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.
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 G Mastoparan can assume an amphipathic 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.
. 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(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 1
, 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 1
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.
-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
-phosphatidyl-D-myo-inositol
4,5-bisphosphate (PI 4,5-bisphosphate) from Roche Molecular
Biochemicals, GDP
S, GTP
S,
L-
-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).
RI
(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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI
) and, perhaps to a lesser extent,
the granule marker (RMCPII) (Fig. 2B). In the same
experiments, ARF-1/GTP
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 GTP S (30 µM) or mastoparan (30 µM) (panel
A) and for cell markers (panel B). The markers were
calnexin (for the endoplasmic reticulum, ER), Fc
RI
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.
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
GTP 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.
-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).
RI). Although the fit was not perfect, the distribution of HA-PLD2 and mastoparan-stimulated activity appeared to best correlate with the distribution of Fc
RI
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).
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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.
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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.
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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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 G
i, and are dependent on PKC or calcium (49, 50). Therefore, mastoparan has
additional activities distinct from its ability to activate G
i; one of them apparently is the ability to activate
PLD2 but not PLD1 in membrane preparations.
-helical conformation on
passing from an aqueous to lipid environment and, by so doing, can
mimic the interactions of physiologic
-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
-helical domains (53), are potent
inhibitors of PLD2 (4, 19), and are present in abundant amounts in
RBL-2H3 membranes.2
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are:
PLD, phospholipase
D;
ARF, ADP-ribosylation factor;
FcRI, 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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Exton, J. H.
(1997)
Physiol. Rev.
77,
303-320 |
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 |
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 |
9. |
Park, S. K.,
Provost, J. J.,
Bae, C. D.,
Ho, W. T.,
and Exton, J. H.
(1997)
J. Biol. Chem.
272,
29263-29271 |
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 |
11. |
Min, D. S.,
Park, S. K.,
and Exton, J. H.
(1998)
J. Biol. Chem.
273,
7044-7051 |
12. |
Bae, C. D.,
Min, D. S.,
Fleming, I. N.,
and Exton, J. H.
(1998)
J. Biol. Chem.
273,
11596-11604 |
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 |
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 |
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 |
18. |
Sung, T. C.,
Altshuller, Y. M.,
Morris, A. J.,
and Frohman, M. A.
(1999)
J. Biol. Chem.
274,
494-502 |
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 |
22. |
Siddhanta, A.,
Backer, J. M.,
and Shields, D.
(2000)
J. Biol. Chem.
275,
1023-12031 |
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 |
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 |
28. |
Cissel, D. S.,
Fraundorfer, P. F.,
and Beaven, M. A.
(1998)
J. Pharmacol. Exp. Ther.
285,
110-118 |
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 |
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 |
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 |
41. |
Dennis, E. A.,
Rhee, S. G.,
Billah, M. M.,
and Hannun, Y. A.
(1991)
FASEB J.
5,
2068-2077 |
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 |
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 |
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 |
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 |
49. |
Chahdi, A.,
Fraundorfer, P. F.,
and Beaven, M. A.
(2000)
J. Pharmacol. Exp. Ther.
292,
122-130 |
50. |
Senyshyn, J.,
Baumgartner, R. A.,
and Beaven, M. A.
(1998)
J. Immunol.
160,
5136-5144 |
51. |
Brown, S.,
Martin, S.,
and Bayley, P.
(1997)
J. Biol. Chem.
272,
3389-3397 |
52. | Boyano-Adanez, M.C., and Gustavsson, L. (2002) Neurochem. Int. 40, 261-268[CrossRef][Medline] [Order article via Infotrieve] |