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INTRODUCTION |
Activation of phospholipase D
(PLD)1 results in the
generation of the lipid second messengers, phosphatidic acid (PA) and
diglycerides, and has been linked to multiple physiologic
processes, including secretion, vesicle trafficking, mitosis, and
meiosis (1-3). In phagocytic leukocytes (monocytes, macrophages, and
neutrophils), activation of PLD is coupled to the major antimicrobial
responses of phagocytosis, generation of reactive oxidants, and granule secretion (1, 4). The mechanisms that regulate PLD and the means by
which its lipid products function in such diverse physiologic processes
are beginning to be elucidated. Two mammalian PLD genes designated PLD1
and PLD2 have recently been cloned (5, 6). PLD1 is regulated by low
molecular weight GTP-binding proteins (GTPases) of the ARF and Rho
families and by protein kinase C (1, 2). The mechanisms that regulate
PLD2 are undefined, but this isoform is unaffected by the activators of
PLD1. The relations between these PLD isoforms and the various PLD
activities demonstrated in diverse cells and tissues requires further study.
Several lines of evidence suggest that PLD may be functionally
associated with the actin-based microfilament cytoskeleton. First, many
of the physiologic processes in which activation of PLD is believed to
play an important role also require rearrangements of the cytoskeleton,
including motility, secretion, and cell division (1, 7-9). This
association of PLD with cytoskeletal-dependent responses is
particularly notable for the primary antimicrobial functions of
phagocytic leukocytes (4, 10-14). Second, stimulation of PLD in
fibroblasts and endothelial cells is coupled to formation of actin
stress fibers, and this effect is mimicked by the addition of purified
PLD or PA and blocked by inhibitors of PLD-dependent generation of PA (15, 16). Third, overexpression of PLD2 promotes cytoskeletal reorganization in serum-stimulated fibroblasts (6). Fourth, several cytoskeletal-associated proteins, including fodrin, synaptojanin, and clathrin-associated protein 3, are potent inhibitors of PLD activity (17-19), suggesting that mechanisms exist for feedback regulation of PLD-dependent cytoskeletal rearrangements.
Fifth, the downstream effectors of PLD modulate actin microfilament
dynamics. PA stimulates the synthesis of
phosphatidylinositol-(4,5)-bisphosphate via activation of
phosphoinositide 4- and 5-kinases (20, 21), and
phosphatidylinositol-(4,5)-bisphosphate, in turn, regulates actin
polymerization (22-24). Diglycerides promote actin nucleation at the
plasma membrane, enhancing the formation of F-actin (25). These
observations suggest a functional association between PLD and the actin
cytoskeleton. The objective of this study was to determine whether
stimulation of GTP-binding proteins results in stable association of
PLD activity with the F-actin-containing membrane skeleton of U937 promonocytes.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise stated, materials were from
previously published sources (26, 27).
[3H]dipalmitoylphosphatidylcholine (DPPC) was obtained
from Amersham Pharmacia Biotech. GTP
S, Gpp(NH)p, and GDP
S were
from Boehringer Mannheim. Phosphatidylethanolamine,
phosphatidylcholine, phosphatidylinositol-(4,5)-bisphosphate, phosphatidyl serine, zymosan, octyl glucoside, sodium cholate, and
molybdenum blue reagent were obtained from Sigma. Silica gel plates
(200 µm) for high performance thin layer chromatography were from
Fisher. Triton X-100 and the reagents for Western blot detection by
enhanced chemiluminescence were obtained from Pierce. PLD1 and PLD2
protein standards were generously provided by Dr. Andrew J. Morris
(State University of New York, Stony Brook, NY).
Antibodies--
Polyclonal antibodies specific for PLD1 or PLD2
were from Quality Controlled Biochemicals Corp. (Hopkington, MA).
Monoclonal Abs to vinculin, talin,
-actinin, and
-COP were from
Sigma, and monoclonal Ab to paxillin was from ICN ImmunoBiologicals
(Costa Mesa, CA). Monoclonal Ab to human major histocompatibility class I was obtained from Dakopatts, Inc. (Denmark). Polyclonal
-RhoA Ab
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and
murine
-ARF was a kind gift of Dr. Richard A. Kahn (National Cancer
Institute, MD).
Cell Fractionation--
U937 promonocytic leukocytes maintained
at 37 °C, 7.5% CO2 in Iscoves medium, 10% fetal bovine
serum, were washed in H/S buffer (25 mM HEPES, pH 7.4, 125 mM NaCl, 0.7 mM MgCl2, 0.5 mM EGTA, 10 mM glucose, 1 mg/ml bovine serum
albumin) (27-29), incubated with 4 mM
diisopropylfluorophosphate, and resuspended in H/K buffer (25 mM HEPES, pH 7.4, 100 mM KCl, 3 mM
NaCl, 5 mM MgCl2, 1 mM EGTA, 2 µM leupeptin, 0.5 mM
phenylmethylsulfonyl-fluoride, 1 mM dithiothreitol) before
disruption by N2 cavitation (450 p.s.i., 25 min). After
removal of undisrupted cells and nuclei by centrifugation at 900 × g, the cavitate was layered over 50% sucrose and
centrifuged at 150,000 × g for 60 min at 4 °C. The
resulting supernatant (cytosol) was re-centrifuged at 225,000 × g and filtered through a 0.2-µm filter. The membrane
fraction at the sucrose interface was pelleted at 225,000 × g for 60 min, resuspended in H/K buffer, and homogenized with a Tenbroeck tissue grinder. This membrane fraction was enriched in
plasma membrane (defined by the presence of virtually all of the human
major histocompatibility class I antigens in a total lysate, data not
shown) and was utilized for all subsequent studies. Because this
fraction also contained the Golgi marker
-COP, it will be referred
to as the "plasma membrane-enriched" or "membrane" fraction,
for simplicity. The more dense fraction, which sedimented through the
50% sucrose, was enriched in the primary granule marker CD63 and was
not utilized in this study. Protein concentrations in membrane and
cytosolic fractions were determined by the method of Bradford (30).
Assay of Membrane Phospholipase D Activity--
75 µg of the
membrane fraction was incubated with 100 µg of cytosol ±GTP
S
(1-100 µM) for 30 min at 37 °C in H/K buffer in a
volume of 100 µl. Membranes were re-isolated by centrifugation at
150,000 × g for 60 min, washed twice with H/K, and
resuspended in same (29). Washed membranes prepared from incubation
with cytosol in the absence of GTP
S are designated Mo,
whereas those prepared in the presence of GTP
S are referred to as
MGTP
S. Substrate vesicles containing
phosphatidylethanolamine:phosphatidylinositol-(4,5)-bisphosphate:phosphatidylcholine (molar ratio of 16:1.4:1) with 10 µCi/sample
[3H]DPPC were prepared by sonication for 10 min at
25 °C (31). 100 µM GTP
S was included in each
sample, and 1.5% ethanol was added to permit detection of the
PLD-specific transphosphatidylation product, phosphatidylethanol (PEt).
Reactions were terminated at 60 min by the addition of 500 µl of
chloroform:methanol (2:1, v/v). Lipids were extracted, dried under
N2, and analyzed by thin layer chromatography (TLC) in an
ethyl acetate:isooctane:acetic acid (9:5:2, v/v) solvent system (26,
27, 29). PEt and PA were identified by comigration with purified
standards, [3H]PEt and [3H]PA cpm were
quantitated by liquid scintillation spectrophotometry, and counts were
normalized for the total amount of 3H-labeled phospholipid
in each experiment. 3H cpm co-migrating with PEt were
determined for each set of samples in the absence of ethanol, and these
background counts were subtracted from each data point.
Phospholipase D Activity of the Detergent-insoluble
Fraction--
Moand MGTP
S, prepared as
noted above, were incubated with 0.5% octyl glucoside in H/K for 30 min on ice. The detergent-insoluble pellets were separated by
centrifugation at 14,000 × g for 15 min at 4 °C,
washed twice in H/K with 0.5% octyl glucoside, and resuspended in H/K
buffer without detergent. The detergent-insoluble fraction prepared
from Mo is designated DIF0, whereas that
derived from MGTP
S is referred to as
DIFGTP
S. The PLD activities of DIFGTP
S
and DIF0 were assayed with [3H]DPPC vesicles,
as noted above, followed by quantitation of [3H]PEt and
[3H]PA. DIFs were also prepared from MGTP
S
and Mo by extraction with 1% sodium cholate or 1% Triton
X-100, and the PLD activities associated with these detergent-insoluble
preparations were determined in an identical manner. In separate
experiments, the direct effects of each detergent on
membrane-associated PLD activity were assessed by incubation of 100 µg of MGTP
S with varying detergent concentrations (0.02-2.0%) in H/K buffer at 37 °C for 60 min, followed by
quantitation of [3H]PEt.
In experiments utilizing C3 exotoxin from Clostridium
botulinum, freshly prepared membrane and cytosol were incubated in
H/K buffer with 1 µg/ml C3 exotoxin, 20 µM nicotinamide
adenine dinucleotide (NAD), 5 mM thymidine for 30 min at
30 °C, as described (32, 33). After the 30-min incubation, 100 µM GTP
S or an equivalent volume of buffer was added,
and the resultant membrane (Mo, MGTP
S, MGTP
S + C3) and detergent-insoluble fractions (DIF0, DIFGTP
S, DIFGTP
S + C3)
were prepared and analyzed for PLD activity. Control incubations were
conducted both in the absence of C3 exotoxin alone, as well as without
C3, NAD, and thymidine, and both sets of controls showed very similar results.
Determination of PLD Activity of DIF Prepared from Permeabilized
U937 Cells--
108 U937 cells (107/ml) in H/K
buffer were incubated with 75 µg/ml
-escin in the presence or
absence of 100 µM GTP
S for 15 min at 37 °C (29).
Permeabilized cell preparations were pelleted at 14,000 × g for 15 min at 4 °C, washed in H/K buffer, and subjected to N2 cavitation under the conditions noted above. Plasma
membrane-enriched fractions were prepared by ultracentrifugation of the
cavitate over 50% sucrose. After washing in H/K buffer, membranes were incubated with 0.5% octyl glucoside for 30 min at 4 °C. The
detergent-insoluble fractions were isolated by centrifugation, washed,
and assayed for PLD activity with [3H]DPPC vesicles.
PLD Activity of DIF Prepared from Intact U937
Cells--
108 U937 cells (107/ml) in Iscoves
medium, 10% fetal bovine serum were washed and resuspended in H/S
buffer. Zymosan was opsonized in 25% serum, as described previously
(12), and incubated with U937 cells for 15 min at 37 °C at a
particle/cell ratio of 10:1. Incubations were terminated by
centrifugation at 3,000 × g for 1 min at 4 °C. The
cell pellet was suspended in H/K buffer then disrupted by
N2 cavitation, and membrane and cytosolic fractions were
prepared as described above. The PLD activity of membrane and octyl
glucoside-insoluble fractions from cells incubated with complement-opsonized zymosan (COZ), designated MCOZ and
DIFCOZ, respectively, were compared with those derived from
cells treated with buffer alone (M0, DIF0) by
determination of [3H]PEt production from
[3H]DPPC vesicles.
Analysis of Phospholipid Content of Membrane and
Detergent-insoluble Fractions--
Lipid phosphorus content of
membrane and detergent-insoluble fractions were determined by the
ashing procedure of Ames (34). Briefly, samples were ashed in
Mg(NO3)2 in ethanol, solubilized in 0.5 M HCl, and heated at 100 °C for 15 min. After cooling to 25 °C, Ames colorimetric reagent (34) was added, samples were incubated at 37 °C for 1 h then cooled to 25 °C, and
A820 nm was determined. Quantitation was
performed by reference to a standard curve derived from
KH2PO4 solutions of known concentration.
SDS-PAGE and Western Blot Analysis--
Membrane, cytosolic, and
detergent-insoluble fractions from U937 cells were prepared as above.
5 × 106 cell equivalents of the membrane or DIF or
100 µg of cytosol were subjected to SDS-PAGE as described previously
(27). 12.5% gels were utilized for analysis of RhoA and ARF, whereas
9% gels were used for detection of PLD, vinculin, talin,
-actinin,
paxillin, and actin. Proteins were transferred to polyvinylidene
difluoride membrane and blocked with 5% nonfat dry milk. Western
blotting, with detection via horseradish peroxidase-coupled 2° Ab and
enhanced chemiluminescence (ECL) was performed as described (27).
Analysis of Data--
Data from each experimental group were
subjected to an analysis of normality and variance. Differences between
experimental groups composed of normally distributed data were analyzed
for statistical significance using Student's t test.
Nonparametric evaluation of other data sets was performed with the
Mann-Whitney Rank Sum test (12).
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RESULTS |
GTP
S Induces a Stable Association of PLD Activity with the
Plasma Membrane and Its Detergent-insoluble Fraction--
We utilized
a cell-free system from U937 promonocytic leukocytes to study
GTP-binding protein-dependent stimulation of PLD activity.
Undifferentiated U937 cells were disrupted by nitrogen cavitation and
subjected to sucrose density centrifugation as described under
"Experimental Procedures." Plasma membrane-enriched and cytosolic
fractions were incubated in the presence or absence of GTP
S,
followed by re-isolation and washing of the membrane fraction. The PLD
activity of these membranes was then determined in the presence of
GTP
S by quantitation of [3H]PEt formation from
DPPC-labeled mixed lipid vesicles in the presence of 1.5% ethanol
(31). Membranes that had been incubated with cytosol in the absence of
GTP
S (Mo) exhibited little basal or
GTP
S-dependent PLD activity upon subsequent isolation
(Fig. 1A). In contrast,
incubation of membrane and cytosolic fractions in the presence of
GTP
S resulted in stable association of PLD activity with the
re-isolated, washed membrane fraction (MGTP
S), which was
21-fold greater than that of Mo. We have previously reported a similar GTP
S-dependent membrane PLD activity
utilizing U937 cells labeled in endogenous lipids with
[3H]oleic acid (29). The PLD activity of
MGTP
S closely approximated the maximal
GTP
S-dependent PLD activity in permeabilized U937 cells
or in the complete cell-free system of membranes + cytosol (29).

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Fig. 1.
GTP S induces stable association of PLD
activity with the plasma membrane and octyl glucoside-insoluble
fraction. A, plasma membrane-enriched (75 µg) and
cytosolic fractions (100 µg) from U937 cells were incubated in the
absence or presence of 100 µM GTP S for 30 min at
37 °C. Membranes (Mo and MGTP S,
respectively) were re-isolated by centrifugation, washed, and assayed
for PLD activity in the presence of 100 µM GTP S and
1.5% ethanol via production of [3H]PEt over 60 min, as
noted under "Experimental Procedures." B, Mo
and MGTP S were incubated with 0.5% octyl glucoside for
30 min on ice. The detergent-insoluble fractions, DIF0 and
DIFGTP S, respectively, were washed twice in H/K buffer
containing octyl glucoside and resuspended in H/K buffer without
detergent for determination of PLD activity in the presence of 100 µM GTP S. PLD activity is expressed as
[3H]PEt cpm/105 [3H]cpm in
phospholipid (PL). Data represent the mean ±S.E. of 15 experiments performed in duplicate.
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We hypothesized that this GTP
S-dependent association of
PLD activity with the membrane fraction involves an interaction with the actin-based membrane skeleton. Mo and
MGTP
S were subjected to extraction with 0.5% octyl
glucoside, and the detergent-insoluble fractions were washed twice in
octyl glucoside-containing buffer, resuspended in detergent-free
buffer, and then assayed for PLD activity in the presence of 100 µM GTP
S. The detergent-insoluble fraction from
Mo (DIF0) exhibited very little PLD activity in response to GTP
S (Fig. 1B). In contrast, the
detergent-insoluble fraction from MGTP
S
(DIFGTP
S) demonstrated significant PLD activity, which
was 20-fold greater than that of control (DIF0). The PLD
activity of DIFGTP
S accounted for approximately 60% of
the total GTP
S-dependent PLD activity of the membrane
fraction (MGTP
S). In contrast to DIFGTP
S,
the octyl glucoside-soluble fraction from MGTP
S
exhibited relatively little PLD activity when assayed versus
[3H]DPPC-labeled vesicles, which accounted for only 17%
of the total PLD activity of MGTP
S (range 15-19%,
n = 3).
Incubation of MGTP
S and DIFGTP
S with
[3H]DPPC vesicles in the absence of ethanol resulted in
significant production of [3H]PA compared with
Mo and DIF0, respectively ([3H]PA
cpm/105 3H cpm in phospholipid:
MGTP
S 5176 ± 548, Mo 265 ± 21, p < 0.001; DIFGTP
S 2478 ± 123, DIF0 58 ± 8 3H cpm, p < 0.001, n = 5 for each). To confirm that PA generation by MGTP
S and DIF GTP
S was because of PLD,
membrane and octyl glucoside-insoluble fractions were incubated in
various concentrations of ethanol, and PA and PEt levels were
determined. Both MGTP
S and DIFGTP
S
demonstrated inverse production of PA and PEt, which was dependent on
the concentration of ethanol (0
1.5%, v/v), as predicted by the
transphosphatidylation capacity of PLD (data not shown). Analysis of
the kinetics of PLD activity of MGTP
S and
DIFGTP
S demonstrated generation of PEt within 1 min of
the addition of ethanol and linear production of PEt throughout the
60-min course of the assay (data not shown).
Stimulation of Permeabilized or Intact U937 Cells Also Results in
Stable Association of PLD Activity with the Detergent-insoluble
Fraction--
To determine whether GTP
S induces the association of
PLD with the DIF in a more complex experimental system, we utilized U937 cells permeabilized with
-escin (26, 29). U937 cells in H/K
buffer were incubated with 75 µg/ml
-escin in the presence or
absence of 100 µM GTP
S for 15 min at 37 °C.
Permeabilized cell preparations were pelleted by centrifugation then
washed in buffer followed by N2 cavitation and isolation of
membranes. Preparation of the octyl glucoside-insoluble fractions was
performed exactly as for the cell-free system, described above. The
octyl glucoside-insoluble fraction from permeabilized cells treated with GTP
S (DIFPGTP
S) contained 2.2-fold greater PLD
activity than the corresponding detergent-insoluble fraction from
control cells incubated with
-escin alone (DIFP0) (range
2.0-2.3, p < 0.01, Fig.
2A). The PLD activity of
DIFPGTP
S accounted for approximately 45% of the total
PLD activity of the plasma membrane fraction from permeabilized cells,
MPGTP
S. These results indicate that activation of
GTP-binding proteins in permeabilized cells results in elevated PLD
activity in the DIF, similar to that demonstrated in the cell-free
system. The decreased PLD activity of DIFPGTP
S (632 ± 47 3H cpm) compared with the DIF derived from the
cell-free system, DIFGTP
S (3263 ± 329 3H cpm) may be because of several factors, including
decreased activation of GTP-binding proteins, decreased access of PLD
to the exogenous substrate vesicles, and/or inclusion of a second detergent (
-escin), which may directly decrease PLD activity.

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Fig. 2.
Stimulation of PLD activity in permeabilized
or intact U937 cells is accompanied by its stable association with
membrane and detergent-insoluble fractions. A, U937
cells (2 × 108) in H/K buffer were incubated with
-escin (75 µg/ml) in the presence or absence of 100 µM GTP S for 15 min at 37 °C. After washing of the
permeabilized cell preparations, samples were subjected to
N2 cavitation, and membranes (MPo and
MPGTP S) were isolated by centrifugation. The octyl
glucoside-insoluble fractions (DIFP0 and
DIFPGTP S) were prepared as noted in the legend to Fig.
1. The PLD activity of each fraction was determined by the formation of
[3H]PEt in the presence of 0.5% ethanol, utilizing mixed
lipid vesicles labeled with [3H]DPPC. B, U937
cells (2 × 108) were incubated with COZ (at a
particle/cell ratio of 10:1) or H/S buffer control for 15 min at
37 °C. Membrane fractions from control (M0) or
COZ-treated cells (MCOZ) were extracted with 0.5% octyl
glucoside, and the detergent-insoluble fractions (DIF0 and
DIFCOZ, respectively) were isolated, washed, and
resuspended in H/K buffer. The PLD activity of each fraction was
assayed as noted in panel A, above. Results represent the
mean ±S.E. of duplicate determinations from three identical
experiments. PL, phospholipid.
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To further evaluate the potential physiologic relevance of
DIF-associated PLD activity, we determined the effects of stimulation of intact U937 cells with a receptor-dependent agonist,
COZ. U937 cells were incubated with COZ (at a particle/cell ratio of
10:1) or H/S buffer control for 15 min at 37 °C. Incubations were
terminated by sedimenting the cells at 3000 × g for 1 min at 4 °C, followed by resuspension in H/K buffer and disruption
by N2 cavitation. Membrane fractions from control
(M0) or COZ-treated cells (MCOZ) were extracted
with 0.5% octyl glucoside, and the detergent-insoluble fractions
(DIF0 and DIFCOZ, respectively) were isolated,
washed, and resuspended in H/K buffer. The PLD activity of
DIFCOZ was 2.0-fold greater than the octyl
glucoside-insoluble fraction from control cells, DIF0
(range 1.9-2.2, p < 0.01, Fig. 2B).
Therefore, a physiologic stimulus, COZ, that binds to complement
receptors on the plasma membrane resulted in stable association of PLD
activity with the DIF, which was qualitatively similar to that produced by GTP
S in permeabilized cells or in the cell-free system. The level
of PLD activity of DIFCOZ (437 ± 32 3H
cpm) approximated that of DIFPGTP
S (derived from
permeabilized cells, 632 ± 47 3H cpm). Because the
cell-free assay provided the greatest level of DIF-associated PLD
activity and was most accessible to experimental manipulations, further
characterization of the association of PLD activity with the
detergent-insoluble fraction was performed with this system.
Analysis of the Protein and Phospholipid Content of Membrane and
Octyl Glucoside-insoluble Fractions--
To serve as a basis for more
detailed characterization of the GTP
S-dependent
association of PLD activity with the membrane and detergent-insoluble
fractions, we determined the chemical composition of these fractions.
The protein contents of M, M0, and MGTP
S
were very similar (Table I). After
extraction with 0.5% octyl glucoside, approximately 35-40% of the
protein content of M0 and MGTP
S remained in
the detergent-insoluble fractions, DIF0 and
DIFGTP
S. In contrast to their similar protein levels,
the membrane and octyl glucoside-insoluble fractions contained
significantly different levels of phospholipid, measured as nmol of
lipid phosphorus via the ashing procedure of Ames (34). The inclusion
of GTP
S in the initial incubation of membrane and cytosol resulted
in an 18% increase in lipid phosphorus in MGTP
S compared with M0 (p < 0.01, n = 3) (Table I). We hypothesize that this
GTP
S-dependent increase in membrane phospholipid content was because of stimulation of de novo synthesis, because the
lipid phosphorus content of MGTP
S exceeded that of the
freshly isolated membrane fraction, M, although this has not been
tested directly. A similar 26% increase in lipid phosphorus occurred in DIFGTP
S compared with DIF0
(p < 0.01).
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Table I
Phospholipid and protein content of membrane and octyl
glucoside-insoluble fractions
75 µg of membrane (M) and 100 µg of cytosol from U937 cells were
incubated in the absence or presence of 100 µM GTP S
for 30 min at 37 °C, followed by washing and re-isolation of the
membrane fractions, Mo, and MGTP S, respectively.
Membranes were extracted with 0.5% octyl glucoside for 30 min at
4 °C, and the detergent-insoluble fractions, DIFo and
DIFGTP S, were isolated, washed, and suspended in H/K buffer.
For each fraction, parallel samples were subjected to analyses of lipid
phosphorus and protein content. For analysis of lipid phosphorus,
samples were solubilized in 0.5 N HCl, followed by the
addition of the Ames colorimetric reagent (34) and determination of
A820 nm. Quantitation was performed by reference to
a standard curve derived from KH2PO4 solutions of known
concentration. Protein levels were determined by the Bradford method
(30). Results are the mean ± S.E. of duplicate determinations from
three identical experiments.
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It is notable that extraction with 0.5% octyl glucoside resulted in
relatively small decreases in phospholipid content in the DIFs compared
with the membrane fractions from which they were derived, suggesting
that this detergent concentration resulted in only modest disruption of
membrane structure. Utilizing both biochemical and electron microscopic
methods, Hartwig et al., (35) demonstrated similar effects
of low concentrations of octyl glucoside on the structure of the
platelet plasma membrane and submembranous actin cytoskeleton.
Concentrations of octyl glucoside
1.0% resulted in extraction of a
significantly greater fraction of phospholipid from membranes of U937
cells (data not shown).
The specific activity of MGTP
S, 331 ± 20.2 [3H]PEt cpm/µg of protein/h, was 20-fold greater than
that of M0, 16.1 ± 1.21 (p < 0.001, n = 3). Because octyl glucoside has a direct inhibitory effect on PLD activity (detailed below), it is not possible to compare
the specific activities of the membrane and detergent-insoluble fractions. If one assumes similar concentrations of octyl glucoside remain in DIFGTP
S and DIF0 after
resuspension in detergent-free buffer, the specific activity of
DIFGTP
S, 515 ± 47.3 [3H]PEt cpm/µg
of protein/h was 22-fold greater than that of DIF0, 23.8 ± 3.40 (p < 0.001, n = 3).
Detergent-dependence of the PLD Activity of
DIFGTP
S--
We evaluated the effect of incubating
MGTP
S in various concentrations of octyl glucoside on
the PLD activity of the resultant detergent-insoluble fractions (after
resuspension in detergent-free buffer). The level of PLD activity
associated with the DIF was inversely proportional to the octyl
glucoside concentration used for extraction of the membrane over the
range 0.1-1.0% (3.4-34 mM). The 0.1% octyl
glucoside-insoluble fraction exhibited a level of PLD activity that was
91% (range 87-95%) that of the total PLD activity of the activated
membrane fraction, MGTP
S, whereas extraction with 1%
octyl glucoside resulted in retention of 42% (range 36-46%) of the
PLD activity in the DIF. It is notable that significant levels of PLD
activity were associated with the detergent-insoluble fraction when the
concentration of octyl glucoside was either below or above the critical
micellar concentration of 20-25 mM (0.59-0.74% octyl glucoside).
To evaluate whether the significant level of PLD activity associated
with the octyl glucoside-insoluble fraction was due, in part, to a
direct enhancement of PLD activity by the detergent itself, we
determined the effect of adding various concentrations of octyl
glucoside to the PLD assay. Octyl glucoside produced dose-dependent inhibition of the PLD activity of
MGTP
S, ranging from a 13 ± 3% reduction at 0.02%
octyl glucoside to a 92 ± 2% decrease in PLD activity at a
detergent concentration of 2.0%. These results are in agreement with
previous reports of detergent-induced inhibition of
GTP
S-dependent PLD activity (1-3). Although we cannot
determine the exact concentration of residual octyl glucoside, which
remains associated with DIFGTP
S, its direct inhibitory effect on PLD activity strongly suggests that the significant levels of
DIF-associated PLD activity presented in Figs. 1 and 2 do not represent
artifactual detergent-mediated enhancement of lipase activity.
To determine whether the GTP
S-dependent association of
PLD activity with the DIF was specific to the use of octyl glucoside, similar analyses were conducted with sodium cholate (critical micellar
concentration 9-15 mM) and Triton X-100 (critical micellar concentration 0.2-0.3 mM). Extraction of
MGTP
S with 1% sodium cholate (23 mM) under
conditions identical to those employed with octyl glucoside resulted in
a 15.2-fold increase in PLD activity in the cholate-insoluble fraction
compared with that obtained from extraction of control membranes
(Mo) when both were assayed in the presence of 100 µM GTP
S (range 14.3-16.1-fold, p < 0.001, n = 3). The PLD activity of the
cholate-insoluble fraction represented 31% (range 28-34%) of the
total PLD activity of MGTP
S. Use of 1% Triton X-100 (15 mM) also resulted in elevated PLD activity in the DIF
fraction derived from MGTP
S (3.2-fold increase over
control, range 1.4-6.4, p < 0.01, n = 4). However, only 2.2% of the original PLD activity of
MGTP
S was retained in the Triton X-100-insoluble
fraction. This low level of Triton X-100-insoluble PLD activity was
likely because of the significant inhibitory effect of this detergent
on PLD activity. In fact, of the detergents tested, Triton X-100
exerted the greatest inhibitory effect on MGTP
S when
added directly to the PLD assay (data not shown). Because the actual
amount of each detergent remaining in the DIF is unknown, it is not
possible to directly compare the PLD activities of the various DIFs
resulting from extraction with octyl glucoside, cholate, or Triton
X-100. However, these results clearly demonstrate that the
GTP
S-dependent association of PLD activity with the detergent-insoluble fraction is a general property exhibited by detergents with significantly different chemical structures (both nonionic and ionic) and critical micellar concentrations.
Guanine Nucleotide-dependence of Detergent-insoluble PLD
Activity--
Membrane and cytosolic fractions were incubated in the
presence of various concentrations of GTP
S (1.0-100
µM), followed by re-isolation of membranes, extraction
with 0.5% octyl glucoside, and determination of PLD activity in the
presence of 100 µM GTP
S. The PLD activities of both
the re-isolated, washed membrane (MGTP
S) and octyl
glucoside-insoluble (DIFGTP
S) fractions were directly proportional to the concentration of GTP
S, which was present in the
initial incubation with cytosol (Fig.
3A). The PLD activity associated with the DIF represented 26, 37, and 54% of the total PLD
activity of the membrane fraction resulting from incubations with
cytosol in the presence of 1, 10, and 100 µM GTP
S,
respectively.

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Fig. 3.
Guanine nucleotide dependence of membrane-
and DIF-associated PLD activity. Membrane and cytosolic fractions
were incubated with the indicated concentrations of GTP S (1-100
µM) (A) or various guanine nucleotide
analogues (B) for 30 min at 37 °C (Gpp(NH)p and
GTP S = 100 µM, GDP S = 1 mM).
After re-isolation and washing of membranes and extraction with 0.5%
octyl glucoside, the PLD activity of the membrane (filled
bars) and DIFs (empty bars) were determined in the
presence of 100 µM GTP S. Data represent the mean
±S.E. of duplicate determinations from three identical experiments.
PL, phospholipid; Ctr, control.
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Further evidence for the GTP-binding protein dependence of the
association of PLD activity with the DIF was obtained with the use of
another nonhydrolyzable analogue of GTP, Gpp(NH)p. Substitution of
Gpp(NH)p for GTP
S in the initial incubation of membrane and cytosol
also resulted in increased PLD activity in the membrane
(MGpp(NH)p)and detergent-insoluble
(DIFGpp(NH)p) fractions, compared with Mo and
DIF0 (Fig. 3B). Gpp(NH)p was less efficacious
than GTP
S in promoting stable association of PLD activity with the
membrane and DIF, which parallels the decreased efficacy of Gpp(NH)p,
relative to GTP
S, in direct stimulation of PLD activity in
permeabilized or cell-free preparations from phagocytic leukocytes (27,
28, 36).
The GDP analogue GDP
S, which cannot be phosphorylated to the
triphosphate form, inhibits GTP-binding protein-mediated responses. Inclusion of a 10-fold molar excess of GDP
S in the initial
incubation of membrane and cytosol resulted in significant inhibition
of GTP
S-dependent, DIF-associated PLD activity (56%
reduction, range 51-62%, p < 0.001, Fig.
3B). The incomplete inhibition of GTP
S-stimulated PLD
activity by GDP
S is consistent with the known biochemical properties
of these guanine nucleotide analogues. GDP
S inhibits the stimulation
of GTP-binding proteins via competition with guanine nucleoside
triphosphates for binding to the nucleotide-free form of the GTPase.
Although GDP
S cannot be phosphorylated to its corresponding
triphosphate, it can be displaced from the GTP-binding protein by GTP
or GTP
S. In contrast, because release of guanine nucleoside
triphosphates requires their hydrolysis, nonhydrolyzable analogues such
as GTP
S are more stably bound to the GTPase than GDP
S. Therefore,
GTP
S-induced responses are only partially inhibited by GDP
S (27,
28, 36). In summary, the studies with guanine nucleotide analogues
strongly support the hypothesis that activation of a GTP-binding
protein(s) mediates stable association of PLD activity with the
detergent-insoluble fraction derived from the plasma membrane.
Effects of GTP
S on the Association of RhoA and ARF with the
DIF--
To test the hypothesis that GTP
S-dependent
association of PLD activity with the DIF is accompanied by the stable
co-localization of RhoA and/or ARF, the levels of these GTPases in
membranes and DIFs were determined by Western blotting. In subcellular
fractions from resting U937 cells (control), RhoA and ARF are present
in both cytosol and membranes, with the majority of each located in
cytosol (Fig. 4A). A
significant fraction of RhoA and, to a lesser extent, ARF, were
extracted from control membranes by 0.5% octyl glucoside (compare M
and DIF0 in Fig. 4A). GTP
S induced concentration-dependent increases in the levels of RhoA and
ARF in membrane (MGTP
S) and octyl glucoside-insoluble
(DIFGTP
S) fractions. Previous work has demonstrated that
guanine nucleotides induce translocation of Rho and ARF GTPases from
the cytosol to membranes (37, 38). Our results confirm these findings
and extend them by demonstrating that GTP
S-dependent
membrane translocation is accompanied by increased association of RhoA
and ARF with the DIF derived from these membranes. Inclusion of GDP
S
in the initial incubation of membrane and cytosol resulted in
inhibition of the GTP
S-induced association of RhoA and ARF with the
membrane and DIF (Fig. 4B). Of note, SDS-PAGE of the
GDP
S-containing samples demonstrated two species of RhoA, one of
which migrated at the standard position for this GTPase, and a second,
which exhibited slightly decreased mobility. At present, we have no
information on the identity of, or the mechanism responsible for, this
slower-migrating form of RhoA.

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Fig. 4.
Effect of GTP S and GDP S on the
localization of RhoA and ARF to membrane and DIF. A,
plasma membrane (M) and cytosol (C) isolated from
U937 cells by nitrogen cavitation and density gradient centrifugation
were incubated in the presence of the indicated concentrations of
GTP S or buffer to prepare MGTP S (1-100
µM) and Mo. Membrane fractions were extracted
with 0.5% octyl glucoside and the respective detergent-insoluble
fractions (1-100 µM DIFGTP S,
DIF0,) were isolated by centrifugation. 5 × 106 cell equivalents of each sample were subjected to
SDS-PAGE on 12.5% gels, followed by transfer to polyvinylidene
difluoride membranes and Western blotting with polyclonal -RhoA or
monoclonal -ARF Abs. The approximate molecular masses for RhoA and
ARF were 22 and 20 kDa, respectively, based on the migration of protein
standards. B, membrane and cytosol were incubated with the
indicated guanine nucleotide analogues (100 µM GTP S, 1 mM GDP S) or buffer for 30 min at 37 °C followed by
re-isolation and washing of the membrane fractions and preparation of
the octyl glucoside-insoluble fractions. Western blotting for RhoA and
ARF was performed as indicated in panel A, above. Results
are representative of three replicates for each experiment.
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To directly evaluate the hypothesis that RhoA functions in the
GTP
S-dependent association of PLD activity with the DIF,
we utilized C. botulinum C3 exotoxin, which catalyzes the
ADP-ribosylation and inactivation of RhoA, RhoB, and RhoC, but not Rac
or Cdc42 (32, 39). Membrane and cytosolic fractions from resting U937 cells were incubated with 1 µg/ml C3 exotoxin prior to the addition of GTP
S and the subsequent preparation of MGTP
S and
DIFGTP
S. Treatment with C3 exotoxin decreased the levels
of RhoA associated with MGTP
S and DIFGTP
S
without significantly affecting the amount of ARF in these fractions
(Fig. 5A), confirming its specific inactivation of Rho GTPases. C3 exotoxin also inhibited the
level of PLD activity associated with MGTP
S (57%
inhibition, range 52-64%, p < 0.001) and
DIFGTP
S (62% inhibition, range 55-66%,
p < 0.001, Fig. 5B). These results are
consistent with a role for Rho GTPases in regulating the association of
PLD with the membrane fraction and the DIF derived from it.

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Fig. 5.
C. botulinum C3 exotoxin reduces the
level of Rho A and the PLD activity of plasma membrane and DIFs.
A, C3 exotoxin (1 µg/ml) from C. botulinum or
buffer control were incubated with membrane and cytosolic
(C) fractions for 30 min at 30 °C before the addition of
GTP S and incubation at 37 °C for an additional 30 min. The washed
membrane and 0.5% octyl glucoside-insoluble fractions were analyzed by
SDS-PAGE/Western blotting with antibodies to RhoA or ARF, as indicated
in the legend to Fig. 4. B, the PLD activity of the membrane
and octyl glucoside-insoluble fractions prepared in panel A,
above, was determined in the presence of 100 µM GTP S
and 1.5% ethanol by formation of [3H]PEt from
[3H]DPPC-labeled vesicles. Data represent the mean ±S.E.
of four experiments performed in duplicate. PL,
phospholipid.
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Effects of GTP
S on the Membrane and Cytoskeletal Localization of
PLD--
To determine whether GTP
S affected the level of PLD
protein associated with the plasma membrane and DIF, we utilized
polyclonal antibodies to human PLD1 and PLD2. Extracts from Sf9
cells infected with baculovirus constructs expressing human PLD1 or
PLD2 served as positive controls for the Western blot experiments. The
anti-PLD2 Ab detected a single species at approximately 125 kDa in the
appropriate positive control (Fig.
6A, 9th
lane); however no PLD2 was detected in any U937 cell
fraction (data not shown). The anti-PLD1 Western blot of the Sf9
cell positive control (Fig. 6A, 3rd
lane) demonstrated a major immunoreactive species at 93 kDa.
Two proteins, of approximately 95 and 93 kDa, were detected in both
membrane (M, Fig. 6A, 8th lane) and
cytosol (C, 1st lane) from U937 cells by the
anti-PLD1 Ab. Because of its co-migration with the PLD1 positive
control, the 93-kDa protein in U937 cell membrane and cytosol is likely to represent PLD1. The identity of the 95-kDa protein is unknown, but
it may represent an isoform of PLD1 and/or stable (covalent) modifications. For simplicity of discussion, both the 93- and 95-kDa
immunoreactive proteins will be referred to as PLD1 and further
specified by their approximate molecular weight.

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Fig. 6.
Antibody to PLD1 detects two major species in
membrane, cytosol, and detergent-insoluble fractions from U937
cells. 5 × 106 cell equivalents of membrane,
cytosol, and cytoskeletal fractions were analyzed by 9% SDS-PAGE,
followed by Western blotting and detection by horseradish
peroxidase-conjugated 2° Ab/ECL. A, the 1st-8th
lanes were probed with polyclonal Ab to PLD1. The
3rd lane is the positive PLD1 control from
baculovirus-infected Sf9 cells. The 9th
lane contains the PLD2 protein standard and is from a
separate blot probed with polyclonal Ab to PLD2. The designation of the
remaining lanes follows the nomenclature in the text.
B, the concentration-dependence of GTP S-induced
association of PLD1 with membrane and DIFs was determined by Western
blotting 5 × 106 cell equivalents of each fraction
with anti-PLD1 Ab. Data in panels A and B are
from two representative experiments utilizing separate preparations of
membrane and cytosol from a total of three replicates for each set of
conditions.
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Incubation of membrane and cytosolic fractions from U937 cells (in the
absence of GTP
S) for 30 min at 37 °C resulted in a significant
decrease in membrane-associated PLD1
(M0, Fig. 6A, 7th lane) and
its accumulation in the supernatant and membrane-wash fractions (data
not shown). In marked contrast, inclusion of GTP
S in the membrane + cytosol incubation resulted in a significant increase in PLD1 in the
re-isolated, washed membrane, MGTP
S (Fig. 6A,
6th lane). Specifically, GTP
S induced a
concentration-dependent increase in the level of the 95-kDa
PLD1 (but not the 93-kDa species) in MGTP
S, compared
with M0 (Fig. 6B).
The GTP
S-dependent association of PLD1 with the plasma
membrane fraction, MGTP
S, was accompanied by similar
association of this lipase with the octyl glucoside-insoluble fraction
derived from it, DIFGTP
S (Figs. 6A, 4th
lane; 6B, 6th-8th lanes). The 95-kDa
species was the major anti-PLD1-immunoreactive protein present in
DIFGTP
S, and its accumulation was dependent on the
concentration of GTP
S over the 1.0-100 µM range. The
level of the 93-kDa PLD1 in DIFGTP
S did not exhibit a
consistent relation to the concentration of GTP
S. DIF0
contained very little 95-kDa PLD1 and a variable amount of the 93-kDa species.
PLD1 Is Stably Associated with the F-actin-containing Fraction
Derived from MGTP
S--
The results of the previous
studies suggest that stimulation by GTP
S induces the stable
association of PLD1, RhoA, and/or ARF into an active lipase complex
that is localized to the membrane surface via interactions with the
F-actin-based membrane skeleton. To test this hypothesis,
MGTP
S was subjected to a broad range of extraction
conditions that varied with respect to detergent concentration, ionic
strength, and pH, followed by separation of soluble and pellet
fractions by centrifugation at 200,000 × g and Western
blotting for PLD1, RhoA, ARF, and actin. The association of PLD1, RhoA,
and ARF with the pellet containing sedimentable actin (F-actin) was
stable to incubation with NaCl over the concentration range of 125-400
mM (Fig. 7A). At
1.0 M NaCl, the majority of PLD1 was extracted from
MGTP
S and localized to the 10% sucrose layer (data not
shown), whereas RhoA and ARF remained predominantly in the pellet. 0.1 M Na2CO3, which extracts many
peripheral membrane proteins (40), completely solubilized PLD1, as did
1.0 M NaOH. Extraction of MGTP
S with octyl
glucoside (0.5-1.0%) or Triton X-100 (0.2-1.0%) resulted in
retention of the majority of PLD1 in the F-actin-containing pellet
(Fig. 7B). At an octyl glucoside concentration of 0.5%,
most of the RhoA and ARF were similarly localized to the pellet
fraction, whereas 1.0% octyl glucoside and 0.2-1.0% Triton X-100
resulted in solubilization of the majority of these GTPases. These
results demonstrate that GTP
S induces the association of PLD1 with
the F-actin-containing fraction and that this association is stable to
widely varying detergent and salt concentrations. RhoA and ARF are
localized to this PLD1 and F-actin fraction under many, but not all, of
these extraction conditions.

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Fig. 7.
Localization of PLD1, RhoA, ARF, and actin to
the soluble or pellet fraction after extraction of
MGTP S. Plasma membrane (M) and cytosolic
(C) fractions were incubated with 100 µM
GTP S to prepare MGTP S, as described under
"Experimental Procedures." 5 × 106 cell
equivalents of MGTP S were incubated with the designated
buffers for 60 min at 4 °C and then centrifuged at 200,000 × g for 60 min at 4 °C. The supernatants (S)
were removed, subjected to repeat centrifugation to remove residual
insoluble material, and subjected to SDS-PAGE. The pellets
(P) were washed in their respective buffers and then
solubilized in SDS sample buffer before PAGE. The initial Western blots
for actin were stripped and reprobed sequentially with Abs to PLD1,
RhoA, and ARF. In panel A, MGTP S was
incubated in the presence of various concentrations of NaCl and
centrifuged over 10% sucrose, whereas panel B demonstrates
the effects of octyl glucoside and Triton X-100 (in 125 mm NaCl buffer)
on the composition of the supernatant and pellet fractions. The results
are from a single experiment (displayed in two panels to
improve the clarity of presentation) from a total of three experiments
performed with different membrane and cytosol preparations.
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To further evaluate the hypothesis that activation of GTP-binding
proteins results in localization of PLD1 to the membrane skeleton,
plasma membrane and octyl glucoside-insoluble fractions were analyzed
for the presence of the cytoskeletal proteins vinculin,
-actinin,
talin, and paxillin. Of note, each of these cytoskeletal proteins have
been specifically localized to the membrane skeleton in multiple cell
types (24, 41). Freshly prepared plasma membrane (M) contained each of
these cytoskeletal proteins (Fig. 8).
After incubation with cytosol at 37 °C (in the absence of GTP
S),
the re-isolated, washed membrane fraction, M0, contained
very little vinculin. Similarly, vinculin was not detectable in
DIF0, and only trace amounts of talin were present in this
fraction. All four of these cytoskeletal proteins were present in
MGTP
S and DIFGTP
S (Fig. 8). Of note,
GTP
S induced a significant increase in the levels of vinculin and
talin present in the octyl glucoside-insoluble fraction. These results
demonstrate that DIFGTP
S has several properties of the
plasma membrane skeleton, including the presence of F-actin and the
cytoskeletal proteins vinculin,
-actinin, talin, and paxillin.

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Fig. 8.
Determination of the presence of cytoskeletal
proteins in membrane and octyl glucoside-insoluble fractions.
Membrane (M, Mo,
MGTP S) and octyl glucoside-insoluble fractions
(DIFo, DIFGTP S)
were prepared as described under "Experimental Procedures" and
subjected to 9% SDS-PAGE. Western blotting was performed with
monoclonal Abs to vinculin, -actinin, talin, and paxillin, with
detection by horseradish peroxidase-coupled 2° goat anti-murine IgG
Ab/ECL. Results are from a single representative experiment from a
total of three.
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 |
DISCUSSION |
Activation of PLD, like that of other phospholipases, must involve
the stable association of the enzyme as well as required regulatory
proteins and co-factors with the membrane surface (1, 42). The
molecular interactions responsible for stabilization of an active PLD
complex at the lipid bilayer are not fully defined. The objective of
this study was to investigate the hypothesis that activation of PLD by
GTP-binding proteins in a plasma membrane-enriched fraction from U937
promonocytes involves an association of the lipase with the
detergent-insoluble membrane skeleton. Extraction of membranes with
octyl glucoside resulted in significant retention of PLD activity in
the DIF, which also contained F-actin. This DIF-associated PLD activity
accounted for approximately 45-90% of the total PLD activity of the
membrane fraction, dependent on the concentration of octyl glucoside
used for extraction. Similar DIF-associated PLD activities resulted
from detergent extraction of membranes with cholate or Triton X-100.
The physiologic relevance of this observation was demonstrated by
similar enhancement of PLD activity in the membrane-derived DIF after
stimulation of intact U937 cells with COZ. To our knowledge, this is
the first demonstration of an association between PLD activity and the
detergent-insoluble membrane skeleton.
The presence, in the DIF, of several cytoskeletal proteins, including
vinculin, talin, paxillin, and
-actinin, and the co-sedimentation of
PLD1 with F-actin under widely varying extraction conditions indicates
that the DIF has many characteristics of the actin microfilament cytoskeleton. The plasma membrane-enriched fraction prepared by sucrose
density gradient centrifugation also contains Golgi-derived membranes
(43), as evidenced by the presence of the Golgi marker,
-COP (data
not shown). Because the Golgi complex is associated with its own
distinct actin cytoskeletal network (44, 45), we cannot, at present,
distinguish the relative contributions of the plasma membrane and Golgi
complex to the PLD activity studied in this report. The term
"membrane skeleton" has been used to indicate the membrane origin
(plasma membrane or intracellular membrane-bound organelles) of the
detergent-insoluble fraction and to distinguish it from the cytosolic
actin-based cytoskeleton (46-48). In the case of stimulation of intact
cells by the phagocytic particle, COZ (12), it seems reasonable to
hypothesize that a significant fraction of the membrane- and
DIF-associated PLD activity is derived from the plasma membrane,
although this has not been formally demonstrated.
Our attempts to further characterize the association of PLD1 with
F-actin by immunofluorescence or immunoprecipitation have been limited
by the specificity and sensitivity of the currently available Abs to
PLD1 (data not shown). To our knowledge, the only data on the
subcellular localization of PLD derived from immunofluorescence was
based on expression of epitope-tagged PLD1 or PLD2 in fibroblasts (6).
The location of endogenous PLD1 and PLD2 in fibroblasts and other cell
types requires further study. Exton and co-workers (49) and Cockcroft
and co-workers (43, 50) characterize the subcellular distribution of
multiple ARF and RhoA-regulated PLD activities (which are likely to
represent PLD1) and present evidence consistent with
agonist-dependent translocation of PLD between these
compartments (43).
A regulated association of PLD activity with the actin-based membrane
skeleton would be relevant to our understanding of the many physiologic
processes in which stimulation of PLD and cytoskeletal rearrangements
have been linked, including mitosis, meiosis, secretion, and motility
(1, 7-9). In the case of myeloid leukocytes, the primary antimicrobial
responses of phagocytosis, generation of reactive oxidants, and granule
secretion involve the actin cytoskeleton, and each has been strongly
associated with stimulation of PLD (4, 10-14).
Cytoskeletal-localization of PLD activity may result in spatially and
temporally restricted generation of the lipid second messengers PA and
diglycerides, which may be important in regulation of these highly
focal leukocyte responses. The membrane skeleton may serve as a focal
point or scaffold for the coordinate assembly of a multiprotein complex
containing regulatory (RhoA, ARF, protein kinase C) and catalytic (PLD)
components (51, 52). Furthermore, because PA and diglycerides promote
remodeling of the membrane skeleton (15, 16, 25), and conversely,
several cytoskeletal proteins are potent inhibitors of PLD, it is
likely that complex feed-forward and feed-back mechanisms exist for
coordinate regulation of PLD and the actin-based cytoskeleton.
Detergent-insoluble plasma membrane domains, including caveolae and
analogous membrane structures in cells that lack caveolin, have
recently been shown to contain numerous signal-transducing molecules,
including low molecular weight and heterotrimeric GTPases, phospholipase C, and tyrosine kinases (53-55). These specialized membrane domains are also enriched in
glycophosphatidylinositol-anchored proteins, glycolipids, and
cholesterol and have been demonstrated to function in membrane
transport processes (transcytosis, endocytosis) and signal transduction
from cell surface receptors (53, 55, 56). We (data not shown) and
others (53, 57) have been unable to detect caveolin in mononuclear
phagocytes by Western blot. However, the demonstration that 0.5% octyl
glucoside results in solubilization of only a minority of plasma
membrane phospholipid (Table I) would be consistent with the presence
of detergent-insoluble, glycolipid-enriched (DIG
(detergent-insoluble
glycolipid-enriched)) domains in U937 cell plasma
membranes. The potential relations between the DIFs studied in this
report and other detergent-insoluble preparations, including caveolae,
will require further study.
The mechanisms that regulate stable association of PLD activity with
the DIF are incompletely defined. Incubation of membrane and cytosol
with GTP
S induced significant increases in the amounts of RhoA and
ARF present in the detergent-insoluble fraction. Although regulation of
the actin cytoskeleton by Rho GTPases has previously been demonstrated
(58, 59), we believe that this is the first description of guanine
nucleotide-dependent association of ARF with the
detergent-insoluble, F-actin-containing fraction. Inhibition of
DIF-associated PLD activity by C. botulinum C3 exotoxin
suggests that Rho family GTPases function in this process. Because
C3-treated samples retain approximately 50% of the PLD activity of
untreated controls, both Rho-dependent and Rho-independent
pathways may mediate stable association of PLD activity with the
membrane skeleton. Rac, CDC42, ARF, or incompletely inactivated Rho
GTPases may be responsible for stimulation of the residual membrane-
and DIF-associated PLD activity in C3-treated samples. Experiments to
distinguish between these possibilities are currently in progress.
The GTP
S dependence of the DIF-associated PLD activity and the
results of Western blotting with PLD-specific Abs suggest that PLD1 is
responsible, at least in part, for the PLD activity of the DIF.
Furthermore, co-migration with the recombinant standard identifies the
93-kDa immunoreactive protein as PLD1. Although overexpressed, PLD2
localized to the plasma membrane in transfected fibroblasts (6), the
GTP
S dependence and lack of immunoreactivity with the
-PLD2 Ab
indicates that PLD2 is unlikely to account for the membrane- and
DIF-associated PLD activities in this report. The approximate molecular
weight of GTP
S-stimulated PLD activities from mammalian cells,
including PLD1a and PLD1b, have ranged from 95 to 120 kDa (51, 52, 60).
Morris and co-workers (52) note the propensity of both PLD1 isoforms to
aggregate during SDS-PAGE (52), which may contribute to this
variability in migration. The identity of the
-PLD1-immunoreactive
95-kDa protein in the membrane and DIF of U937 cells is currently
unknown. PLD1a and PLD1b could account for the 95- and 93-kDa proteins
(because the polyclonal Ab utilized in this study recognizes a shared
peptide sequence). However, this probably would not account for the
GTP
S-dependent increase in the level of the 95-kDa
immunoreactive protein in MGTP
S and
DIFGTP
S (compared with M0 and
DIF0), because the alternative splicing responsible for the
generation of PLD1a and PLD1b would not be expected to occur in the
cell-free system. An alternative explanation for the results of the
anti-PLD1 Western blot is the presence of a single PLD isoform, which
exhibits altered mobility because of covalent modification (60). We
cannot exclude the formal possibility that the 95-kDa protein is a
previously uncharacterized PLD, distinct from PLD1, that is
cross-reactive with the
-PLD1 Ab.
Approximately 15% of the membrane PLD activity was recovered in the
0.5% octyl glucoside-soluble supernatant. This likely represents an
underestimate because of the inhibition of PLD activity by this
concentration of octyl glucoside and because PLD activity was assayed
in the absence of added co-factors such as low molecular weight GTPases
or protein kinase C. Sternweis and co-workers extensively characterize
a GTPase- and protein kinase C stimulated PLD activity in detergent
extracts from membranes of HL-60 cells (31) or porcine brain (51, 61),
which was assayed in the presence of 0.05% octyl glucoside. The
relation between the PLD activity extracted from plasma membranes of
U937 cells and that reported by Sternweis and co-workers will require
further analysis.
In summary, this report demonstrates that stimulation of GTP-binding
proteins or plasma membrane receptors of U937 promonocytes results in
stable association of PLD activity with the detergent-insoluble fraction, which has many characteristics of the actin-based membrane skeleton. We propose that cytoskeletal localization of PLD activity may
promote focal regulation of the actin microfilament system and function
in the control of physiologic processes, which require both stimulation
of PLD and cytoskeletal rearrangements.