From the
Phospholipase D (PLD) activity that was stimulated by guanosine
5`- O-(3-thiotriphosphate) (GTP
Rho-specific GDP dissociation inhibitor inhibited GTP
Phospholipase D (PLD)
Although PLD activity has been
shown to be associated with membranes in mammalian cells
(15, 16, 17, 18, 19) , there is
one report of its existence in the cytosol
(20) . In HL60 cells,
PLD activity was reported in membranes and shown to be regulated by
GTP
The
present investigation of PLD activity in HL60 cells has revealed the
presence of PLD in cytosol as well as membranes. In addition, it has
been found that the membrane-bound PLD can be stimulated by GTP
Soluble ADP ribosylation factor
(sARF) (a mixture of ARF1 and ARF3) was purified from bovine or rat
brain cytosol
(31) . Polyclonal antibodies were raised against
sARF according to Tsai et al. (32) . Both were kind
gifts of S.-C. Tsai and J. Moss (National Institutes of Health).
Antibodies against C-terminal peptides of RhoA, RhoB, Rac1, and Rac2
were from Santa Cruz Biotechnology Corporation. Glutathione
S-transferase-fused Rho-specific GDP dissociation inhibitor
(GST-Rho-GDI) was purified from E. coli transformed with the
relevant plasmid
(30) . This plasmid and antiserum against
CDC42Hs were gifts from Y. Zheng and R. Cerione (Cornell University).
HL60 cells are one of the most extensively studied cell types
with respect to the regulation of PLD activity. It is generally
believed that the PLD activity in these cells is located in the
particulate fraction and that its stimulation by guanine nucleotides is
mediated by cytosolic factors. One of the cytosolic factors has
recently been identified as ARF. Like previous findings, our studies
with [
Our observations are consistent
with the finding that the stimulation of the hydrolysis of exogenous PC
by membrane PLD in the presence of GTP
The use of exogenous substrate
also made it possible to uncover the presence of PLD in the cytosol.
Chromatography of this fraction separated the PLD activity from SMGs.
Although several SMGs were found, the initial co-elution of PLD
activity with ARF immunoreactivity supported the involvement of ARF in
the regulation of cytosolic PLD activity. Further evidence was obtained
when the PLD activity peak from DEAE was separated from the SMGs by two
different chromatographic procedures. In fractions in which PLD was
separated from ARF, activity was restored by either endogenous ARF
(separated on the column) or exogenous ARF (bovine sARF). However, RhoA
and CDC42 were ineffective. It is interesting to note that
GTP
Both membrane and cytosolic PLDs were
GTP
The cytosolic PLD was stimulated by ARF
but not RhoA or CDC42 (which are all present in the cytosol), whereas
the membrane PLD could be stimulated by members of the Rho family
(RhoA, Rac1, and CDC42) and also ARF (Figs. 9 and 11). Previously,
Bowman et al. (27) showed that GTP
To further emphasize the role of
RhoA, we also studied the translocation of Rho proteins from cytosol to
the membranes in the absence and presence of GTP
Although,
RhoA and ARF synergistically stimulate membrane PLD, stimulation by
RhoA does not require the presence of ARF.
In summary, our observations support the results of earlier
investigations into the control of PLD by ARF and Rho
(25, 26, 27) and provide another example of the
specific role of RhoA
(30) . They strongly suggest the existence
of PLD isozymes that differ in cellular location and regulation. The
Rho-regulated isozyme appears to be present only in membranes, while
the ARF-responsive enzyme is found in both cytosol and membranes.
However, since ARF is absent from the isolated membranes, it would have
to be translocated there in order to regulate the enzyme, and this may
be true for Rho also. Clearly, elucidation of the mechanisms of
regulation of PLD by both SMGs will require much further work.
We thank Y. Zheng and R. Cerione (Cornell University)
for kind gifts of the GST-Rho-GDI plasmid and the antiserum against
CDC42Hs, and S.-C. Tsai and J. Moss (National Institutes of Health) for
generous gifts of sARF and sARF antibodies. We also thank J. Childs for
typing this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S) was detected in cytosol
and membranes of HL60 cells. GTP
S-stimulated PLD activity was
detected in the membranes when exogenous labeled phosphatidylcholine
was used in the presence of phosphatidylethanolamine and
phosphatidylinositol 4,5-bisphosphate, but not when
[
H]myristic acid-labeled endogenous substrate was
used. Cytosolic PLD co-chromatographed with small GTP-binding proteins
on anion-exchange columns, but subsequent chromatography separated
these. Reconstitution studies demonstrated ADP ribosylation factor
(ARF) as a regulator of cytosolic PLD, whereas the Rho proteins RhoA
and CDC42Hs were ineffective. The cytosolic enzyme showed very little
activity in the absence of GTP
S and was stimulated by 2 m
M Ca
, whereas the membrane enzyme had significant
basal activity and was inhibited by Ca
.
S
stimulation of membrane PLD activity in the presence and absence of
cytosol. The stimulation in GDP dissociation inhibitor-treated
membranes could be partially recovered by the addition of recombinant
Rho proteins (RhoA, Rac1, CDC42Hs). RhoA and Rac1 were also stimulatory
in untreated membranes. However, Western blot analysis of membranes
showed the presence of RhoA, but not Rac1 or CDC42Hs, suggesting that
RhoA was the endogenous small GTP-binding protein involved in
GTP-dependent PLD activity in membranes in the absence of cytosol. ARF
also stimulated the membrane PLD in the presence of GTP
S, and the
combination of RhoA and ARF showed a synergistic effect. These results
show the presence of ARF-dependent PLD activity in both cytosol and
membranes. The membranes contain another PLD activity for which the
endogenous regulator appears to be RhoA. The data suggest the existence
of at least two different PLD isozymes in HL60 cells.
(
)
catalyzes the
hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and
choline and has been implicated in signal transduction in a variety of
cell types
(1, 2, 3, 4, 5) .
During the last decade, a large number of agonists that stimulate PLD
have been identified
(1, 2, 3, 4, 5) , and PA has
been proposed to regulate DNA synthesis, cell proliferation, and other
cellular functions
(5, 6, 7, 8, 9, 10, 11) .
PA can also be metabolized to diacyglycerol, which in turn stimulates
protein kinase C. A unique property of PLD is its catalysis of the
transphosphatidylation reaction in which the phosphatidyl moeity of
phospholipids is accepted by a primary alcohol
(12, 13, 14) .
S in the presence of a cytosolic factor
(21, 22) , which was proposed to be a G protein
(23, 24) . Two recent reports
(25, 26) have revealed that the cytosolic factor is the small G
protein (SMG) ADP ribosylation factor (ARF). This 21-kDa protein was
first identified and purified on the basis of its activity as a
cofactor for cholera toxin-catalyzed ADP ribosylation of the
-subunit of G
(28, 29) . A recent study
has also suggested the involvement of a membrane-associated G protein
of the Rho family in PLD regulation
(27) . This study
demonstrated that GTP-dependent PLD activity in HL60 cell preparations
was stimulated by a nonspecific GDP dissociation stimulator and
inhibited by a Rho-specific GDP dissociation inhibitor (GDI).
S
in the absence of cytosol, and evidence is presented that the G protein
involved is RhoA.
Materials
Dipalmitoylphosphatidylcholine
(dipalmitoyl-PC), phosphatidylethanolamine (PE), PA, and
phosphatidylethanol (PEth) were purchased from Avanti Polar Lipids
Corp. Phosphatidylinositol 4,5-bisphosphate (PIP) and
GTP
S were obtained from Boehringer Mannheim.
[9,10-
H]Myristic acid, dipalmitoyl
[2- palmitoyl-9,10-
H]phosphatidylcholine,
and
dipalmitoylphosphatidyl[ methyl-
H]choline
were from DuPont NEN. HL60 cells were obtained from ATCC. DEAE Sephacel
and Superdex 200 were from Pharmacia Biotech Inc. Hydroxylapatite was
from CalBiochem.
SMGs and Antisera
The cDNAs of Rac1 and Rho A
(gifts of A. Hall, University College, London) and of CDC42 (a gift of
S. Munemitsu, Onxy Pharmaceuticals) were provided with a modified
Glu-Glu epitope tag at the N terminus, cloned into pAcC13, and
transfected into Sf9 cells to produce recombinant baculovirus, and the
proteins were purified on a Glu-Glu monoclonal antibody affinity column
as described by Malcolm et al. (30) . Since the
proteins were prepared from Sf9 cytosol, it is likely that they were
partly modified by prenylation.
Culture and Labeling of Cells
HL60 cells were
maintained in suspension culture in RPMI 1640 medium (Cell Gro)
containing 10% fetal bovine serum (Sigma), at 37 °C in a humidified
atmosphere of 5% COand 95% air. Cells were harvested when
confluent (1
10
cells/ml). Cells were labeled with
[
H]myristic acid, and membranes were prepared as
described
(21) . Membranes were aliquoted (0.5-1.0 mg
protein/ml) and stored at -80 °C.
Preparation of Membranes and Cytosol
Harvested
HL60 cells (4-6 10
) were washed twice in
buffer. A (25 m
M HEPES (pH 7.2), 125 m
M NaCl, 2.5
m
M KC1, 0.7 m
M MgCl, 0.5 m
M EGTA, 10 m
M glucose). Washed cells were resuspended in buffer B (5 m
M HEPES (pH 7.4), 1 m
M EGTA, 1 m
M dithiothreitol,
250 m
M sucrose, 12 µg/ml leupeptin, 10 µg/ml
aprotinin, 20 µg/ml 4-amidinophenylmethanesulfonyl fluoride, and 20
µg/ml antipain) and were disrupted in a bomb by rapid decompression
after equilibration with N
at 4 °C for 20 min at 500
psi. Unbroken cells and nuclei were removed by centrifugation at 500
g for 10 min. Membranes and cytosol were obtained by
centrifugation at 125,000
g for 90 min. Supernatant
(cytosol) was used for chromatography, whereas the pellet (membranes)
was washed twice in buffer C (50 m
M HEPES (pH 7.2), 10 m
M KCl, 5 m
M NaCl, 0.5 m
M EGTA, and 3.5 m
M MgCl
). Membranes were aliquoted (5-10 mg/ml) and
stored at -80 °C.
Chromatography of HL60 Cytosol
150 ml of cytosol
(1 mg of protein/ml) was loaded on a DEAE-Sephacel column (2.5
12 cm). The column was washed with buffer D (25 m
M HEPES (pH
7.4), 1 m
M EGTA, and 1 m
M dithiothreitol), and the
bound proteins were eluted with a 200-ml linear gradient of NaCl
(0-0.8
M) in buffer D. Fractions (3 ml) were assayed for
PLD activity, and active fractions were pooled and used for further
purification and characterization of PLD. For gel filtration
chromatography, the pool of active fractions was concentrated to 3 ml
using a YM10 Amicon membrane and applied to a 150-ml Superdex 200
column in buffer A. This was eluted with the same buffer. Column
fractions (2 ml) were assayed for PLD activity in the presence and
absence of bovine sARF. Alternatively, a pool of active DEAE fractions
(from another cytosol preparation) was diluted with buffer D (without
EGTA) to decrease the concentration of NaCl to 50 m
M. The
diluted pool was then loaded onto a 10-ml hydroxyapatite column. The
column was extensively washed with buffer E (25 m
M HEPES (pH
7.4), 1 m
M dithiothreitol, and 50 m
M NaCl), and the
adsorbed material was eluted with a linear gradient of potassium
phosphate (0-150 m
M) in buffer E. Fractions (1 ml) were
assayed for PLD activity in presence and absence of sARF.
Assays of PLD
[H]Myristic
acid-labeled HL60 membranes were assayed for PLD activity as described
(21) . PLD activity was measured in unlabeled membranes,
cytosol, and column fractions, using phospholipid
(PE/PIP
/PC, 16:1.4:1) vesicles prepared according to Brown
et al. (25) .
Dipalmitoyl[2- palmitoyl-9-10-
H]PC
(0.5 µCi) (for [
H]PEth formation) or
dipalmitoyl[ methyl-
H]choline PC (0.5
µCi) (for [
H]choline release) was included in
the phospholipid mixture for each assay. For a 60-µl assay volume,
10 µl of vesicles were added to the assay buffer (50 m
M HEPES, (pH 7.2), 3 m
M EGTA, 80 m
M KCl, 1 m
M dithiothreitol, 3 m
M MgCl
, and 2 m
M CaCl
) containing membranes, cytosol, or column
fractions and were incubated at 37 °C for 30 min (unless otherwise
indicated). For the [
H]choline release assay, the
reactions were stopped by the addition of 200 µl of 10%
trichloroacetic acid and 100 µl of 1% bovine serum albumin, and the
mixtures were processed as described
(25) . For
[
H]PEth formation, ethanol (1% (v/v) for
membranes or 2% (v/v) for cytosol and column fractions) was included in
the reaction mixtures, and the reactions were terminated by the
addition of 375 µl of chloroform/methanol/HC1 (50:98:2), and the
lipids were extracted as described
(33) . Separation and Quantitation of [
H]PEth and
[
H]PA-Lipids from the incubation
mixtures were resuspended in chloroform/methanol (2:1) and spotted onto
thin layer plates (LK6D, Silica gel, Whatman). The plates were
developed with ethyl acetate/isooctane/acetic acid/water (50:25:10:50,
modified from Ref. 34). This solvent system gives better separation of
PA and PEth. Separated phospholipids and standards were visualized on
thin-layer chromatography plates by exposure to iodine vapor. Spots
corresponding to PA and PEth were scraped, mixed with ready organic
scintillant (Beckman), and counted.
SDS-Polyacrylamide Gel Electrophoresis and Western
Analysis
SDS-polyacrylamide gel electrophoresis was performed on
14% acrylamide gels or gradient 10-25% acrylamide Novex gels
(Novel Experimental Corp.). For Western analysis, proteins were
transferred from SDS-polyacrylamide gels onto Immobilon P membranes
(Millipore) for 1 h at 15 V using a semi-dry transfer apparatus
(Bio-Rad). The immunoblots were blocked overnight with 1% (w/v) bovine
serum albumin and 1% (v/v) goat serum and incubated for 2 h with
primary antibody in blocking buffer. Blots were developed using the
Vector stain alkaline phosphatase ABC kit (Vector Laboratories,
Burlingame, CA).
Membrane Extraction
HL60 membranes were incubated
with GST-Rho-GDI in buffer F (20 m
M HEPES (pH 7.0), 10 m
M glutathione) at 4 °C for 60 min. After incubation, membranes
were centrifuged at 14,000 g for 10 min. Supernatants
were collected, and membranes were washed twice in solution A. For
controls, membranes were incubated in buffer F in the absence of
GST-Rho-GDI. The supernatants (membrane extracts) and membranes were
analyzed by Western blotting using antibodies against small G proteins
(RhoA, RhoB, CDC42, and Rac1).
Distribution of PLD in HL60 Cells
Guanine
nucleotide-sensitive PLD activity was detected in both membranes and
cytosol of HL60 cells (Fig. 1 A). In contrast to previous
reports
(25, 26, 27) significant GTPS
stimulation of PLD activity was observed in the membranes in the
absence of cytosol, but only when exogenous
[
H]dipalmitoyl-PC was employed as substrate
together with PE and PIP
in the molar ratio described by
Brown et al. (25) . In the absence of PIP
,
the activity was very low (data not shown, see Ref. 25). The
stimulation was enhanced by the addition of cytosol (Fig. 1 A).
In contrast, [
H]myristic acid prelabeled
membranes failed to exhibit GTP
S-sensitive
[
H]PEth formation in the absence of cytosol
(Fig. 1 B). Since oleate has been reported to stimulate
membrane PLD in some cell types
(5) , its effects were tested on
HL60 membranes using exogenous [
H]dipalmitoyl-PC,
but no stimulation was observed (data not shown).
Figure 1:
GTPS stimulation of PLD activity
in HL60 membranes and cytosol. Panel A, HL60
membranes and cytosol were prepared as described under
``Experimental Procedures.'' HL60 membranes (30 µg of
protein) or cytosol (10 µg of protein) or membranes plus cytosol
were incubated for 30 min in the presence and absence of 30 µ
M GTP
S as indicated. [
H]PEth formation
from [
H]palmitoyl-PC was measured in the presence
of 1% ethanol under the conditions of Brown et al. (25) as
described under ``Experimental Procedures.'' Panel B, HL60 cells were prelabeled with [
H]myristic
acid, and membranes and cytosol were prepared as described (21).
Labeled membranes (20 µg of protein) or labeled membranes plus
cytosol (10 µg of protein) were assayed for PLD activity using the
conditions described under ``Experimental Procedures.''
Cytosol alone showed no activity. The data shown in panels A and B are representative of two
experiments.
Partial Purification of Cytosolic PLD
To study the
GTPS-sensitive cytosolic PLD activity, HL60 cytosol was
fractionated by DEAE-Sephacel chromatography. A peak of
GTP
S-dependent PLD activity was eluted in the early part of the
NaCl gradient (Fig. 2). In order to investigate the involvement of
SMGs, column fractions were also immunoblotted using anti-ARF,
anti-RhoA, and anti-CDC42Hs antibodies. As shown in Fig. 2, these
SMGs were found in the fractions containing PLD activity. However, ARF
immunoreactivity corresponded to the major fractions exhibiting
GTP
S-dependent PLD activity, although there was a minor component
of PLD activity (fractions 30-34) that coeluted with RhoA and
CDC42. In addition, we assayed the remaining inactive fractions in the
presence of bovine cytosolic ARF (sARF) and detected no ARF-responsive
enzyme. The fractions corresponding to the peak of GTP
S-stimulated
PLD activity were pooled and concentrated using an Amicon YM 10
membrane.
Figure 2:
Fractionation of HL60 cytosol on
DEAE-Sephacel. Panel A, HL60 cytosol was obtained as
described under ``Experimental Procedures.'' It was
chromatographed on DEAE-Sephacel, and the fractions were assayed for
PLD activity. An aliquot (10 µl) of every second fraction was
incubated with 30 µ
M GTPS for 30 min, and
[
H]choline release from
phosphatidyl[
H]choline was measured as described
under ``Experimental Procedures.'' Panel B, an
aliquot (10 µl) of every second fraction was diluted with an equal
volume of Laemmli SDS sample buffer, and 5 µl of this was subjected
to electrophoresis using 14% polyacrylamide gels. After transfer to
Immobilon P, proteins were blotted using antisera specific for ARF,
RhoA and CDC42Hs.
To determine the molecular size of the ARF-responsive PLD,
the concentrated pool was further fractionated by gel filtration using
a Superdex 200 column. A lesser peak of GTPS-stimulated PLD
activity was observed in the fractions that contained ARF (Fig. 3).
Assuming that PLD activity may have separated from endogenous ARF, we
assayed the remaining fractions in the presence of sARF. This revealed
a peak of GTP
S-stimulated PLD activity, which corresponded to a
molecular mass of >150 kDa (Fig. 3). The nature of the higher
molecular mass material was not determined, but it was assumed to be
aggregated enzyme.
Figure 3:
Gel filtration chromatography of a
concentrated PLD activity peak from DEAE-sephacel. Panel A, the concentrated pool of PLD active fractions (see
``Experimental Procedures'') was applied to a Superdex 200
column and eluted as described under ``Experimental
Procedures.'' Every second eluted fraction was assayed for
[H]PEth formation from
[
H]palmitoyl-PC with the addition of 30
µ
M GTP
S in the presence ( closed symbols) and absence ( open symbols) of
bovine sARF. Panel B, aliquots (10 µl) of every second
fraction were diluted with an equal volume of Laemmli SDS sample
buffer, and 5 µl of this was subjected to SDS-polyacrylamide gel
electrophoresis and Western blotting as described for Fig.
2 B.
To further explore a role for ARF in the
regulation of cytosolic PLD, we also chromatographed the concentrated
pool of DEAE fractions on a hydroxylapatite column. Fractions from this
column totally failed to show any GTPS-stimulated PLD activity
unless reconstituted with bovine sARF (Fig. 4). The
ARF-responsive PLD activity eluted in the late phase of the phosphate
gradient. Endogenous ARF from the same column also restored the PLD
activity, but no activity was detected with fractions containing
endogenous Rho A or CDC42.
Figure 4:
Hydroxylapatite chromatography of
concentrated PLD activity peak from DEAE sephacel. Panel A, the concentrated pool of PLD active fractions, diluted
with Buffer E as described under ``Experimental Procedures,''
was applied to a hydroxylapatite column, and eluted fractions (every
third) were assayed for [H]PEth formation from
[
H]palmitoyl-PC in the presence of 30 µ
M GTP
S and in the presence (
) or absence (
) of
bovine sARF. Panel B, aliquots (10 µl) of every third
fraction were subjected to electrophoresis and Western blotted as
described in Fig. 2.
Further Characterization of Cytosolic PLD
The
concentrated pool of active PLD fractions from DEAE-Sephacel,
hereinafter referred to as the DEAE fraction, was used for further
characterization of the cytosolic PLD. The time course of PEth
formation in the presence of GTPS was linear for 40 min, but
essentially no activity was seen in the absence of the nucleotide (data
not shown). The time course was similar to that of the
membrane-associated activity (assayed with exogenous
[
H]PC), except that the latter showed significant
activity in the absence of GTP
S (data not shown). The GTP
S
concentration producing half-maximal stimulation of PEth formation by
either cytosol or membranes was less than 1 µ
M (Fig. 5). This is similar to that observed in
cytosol-depleted HL60 cells in the presence of ARF
(26) .
Figure 5:
GTPS dependence of PLD activation in
DEAE fraction and membranes. The DEAE fraction (10 µg of protein)
or membranes (30 µg of protein) was incubated with the indicated
concentrations of GTP
S, and [
H]PEth
formation from [
H]palmitoyl-PC was measured as
described under ``Experimental Procedures.'' Data are
expressed as the percent of total lipid radioactivity recovered in PEth
at the end of the incubations and are representative of two experiments
performed in duplicate.
GTPS-dependent PLD activity in the DEAE fraction was linearly
proportional to protein concentration up to 10 µg (data not shown).
Although millimolar Ca
has been reported to stimulate
PLD activity in HL60 cell membranes assayed using endogenous substrate
(21, 25) , the cation had a negligible stimulatory
effect when the membranes were assayed with exogenous PC, and was
inhibitory at concentrations of 2 m
M and higher
(Fig. 6). In contrast, PLD in the DEAE fraction showed a 2-fold
stimulation with 2 m
M Ca
(corresponding to
165 n
M free Ca
, see the legend to
Fig. 6
), which was lost at higher Ca
concentrations.
Figure 6:
Effect of Ca on PLD
activity in HL60 membranes and DEAE fraction. HL60 membranes (30 µg
of protein) or DEAE fraction (10 µg of protein) were incubated with
the indicated concentrations of CaCl
in the presence of 30
µ
M GTP
S to measure [
H]PEth
formation for 30 min. The incubation medium contained 3 m
M EGTA (see ``Experimental Procedures''), and the
concentrations of free Ca
corresponding to the added
Ca
concentrations were calculated to be 165 n
M for 2 m
M, 8.1 µ
M for 4 m
M, 25.4
µ
M for 6 m
M, and 70.6 µ
M for 10
m
M. Data are representative of two experiments performed in
duplicate.
Identification of the SMGs Regulating Membrane
PLD
In view of a previous report indicating the involvement of
an unidentified Rho family SMG in the regulation of PLD
(27) ,
we utilized HL60 membranes to examine GTPS-stimulated PLD activity
as a function of increasing concentrations of GST-Rho-GDI
(Fig. 7 A). As observed earlier
(27) ,
GTP
S-stimulated membrane PLD activity, assayed using endogenous
substrate ([
H]myristate-labeled membranes) in the
presence of cytosol, was significantly inhibited by micromolar Rho-GDI.
However, when assayed in the absence of cytosol using exogenous
[
H]PC, Rho-GDI was more potent and efficacious in
inhibiting the PLD activity (Fig. 7 B). Since treatment
of membranes with Rho-GDI has been reported to extract
post-translationally modified Rho proteins
(35, 36) ,
Western blotting of the supernatants was performed using antibodies to
RhoA, RhoB, CDC42, Rac1, and Rac2. The results showed that RhoA was
significantly extracted from the membranes, whereas Rac1, Rac2, or
CDC42 were not detected in the extracts (data not shown). The presence
of Rac1, Rac2, and CDC42 was also examined in unextracted HL60
membranes, and these were also found to be absent (data not shown). In
contrast, HL60 cytosol showed the presence of CDC42 and RhoA, but not
Rac1 immunoreactivity ( Fig. 2and data not shown).
Figure 7:
Inhibition of GTPS-stimulated PLD
activity in membranes by Rho GST-GDI. Panel A, HL60
cells were prelabeled with [
H]myristic acid as
described previously (21). The labeled membranes (20 µg of protein)
and cytosol (10 µg of protein) were preincubated with indicated
concentrations of Rho GST-GDI on ice for 15 min. Incubations were
continued at 37 °C for 30 min in the presence of 30 µ
M GTP
S. [
H]PEth formation in the presence
of 1% ethanol was measured as described under ``Experimental
Procedures.'' Panel B, HL60 membranes were
preincubated with the indicated concentrations of Rho GST-GDI in the
absence of cytosol and assayed for [
H]PEth
formation from exogenous [
H]palmitoyl-PC under
the same experimental conditions as in Fig. 1 A. Data for both
experiments are representative of two experiments performed in
duplicate.
The
GDI-extracted membranes were tested for the restoration of
GTPS-dependent PLD activity after reconstitution with recombinant
RhoA, CDC42, and Rac1. All of these SMGs partially restored the PLD
activity in the order of efficacy RhoA > CDC42 > Rac1 (Fig. 8).
In the absence of GTP
S, none of the SMGs was effective (data not
shown). Interestingly, recombinant RhoA and Rac1 were also effective in
a dose-dependent manner on unextracted membranes, whereas CDC42 was
ineffective (Fig. 9). To further explore the role of RhoA, we
also studied the translocation of Rho proteins from cytosol to
membranes in the presence of GTP
S. Although RhoA and CDC42 were
detected in HL60 cytosol, only RhoA was translocated in a
time-dependent manner in the presence of GTP
S (Fig. 10). PLD
activity in unextracted membranes was also tested with bovine sARF, and
a stimulation was observed (Fig. 11), as expected
(25) . The
reconstitution of membranes with a mixture of increasing concentrations
of recombinant RhoA and bovine sARF showed a synergistic effect on PLD
activity, although this was not marked (Fig. 11).
Figure 9:
Effect of recombinant Rho proteins on the
PLD activity of unextracted membranes. HL60 membranes (30 µg) were
preincubated with indicated concentrations of RhoA, CDC42, and Rac1 in
the presence of GTPS (30 µ
M) on ice for 15 min. The
incubations were continued at 37 °C for 30 min in the presence of
1% ethanol. [
H]PEth from
[
H]palmitoyl-PC was quantitated as described
under ``Experimental Procedures.'' A representative
experiment of two performed in duplicate is
shown.
Figure 10:
GTPS-dependent translocation of Rho
A from cytosol to membranes. HL60 membranes (0.1 mg) and cytosol (0.05
mg) were incubated in PLD assay buffer, in the presence and absence of
GTP
S (50 µ
M) at 37 °C for the indicated times.
After incubation, membranes were centrifuged at 14,000
g for 10 min, and pellets were washed twice in buffer A. Pellets
were resuspended in Laemmli SDS sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis and Western blotting as
described under ``Experimental
Procedures.''
Figure 11:
Effect of RhoA and ARF on membrane PLD
stimulation. HL60 membranes (30 µg of protein) were preincubated
with the indicated concentrations of recombinant RhoA or bovine sARF or
a combination of the RhoA and ARF in the presence of GTPS (30
µ
M) on ice for 15 min. The experiment was continued as
described in Fig. 10. Data are representative of two
experiments.
H]myristic acid-labeled cell membranes also
indicate the requirement of cytosol for GTP
S-dependent PLD
activity. However, when we assayed the enzyme with exogenous substrate
according to Brown et al. (25) , surprisingly, we found
GTP
S-dependent activity in the soluble as well as the particulate
fraction. Furthermore, we found that the PLD activity in the
particulate fraction could be stimulated up to 5-fold with GTP
S in
the absence of cytosol. These two interesting observations provoked us
to further investigate the regulation of PLD activity in both
particulate and soluble fractions.
S plus ARF requires the
addition of PIP
(25, 37) . Also, in
agreement with other findings
(25, 26, 27) we
detected significant stimulation of HL60 membrane PLD by GTP
S plus
cytosol or plus bovine sARF when [
H]myristic
acid-labeled membranes were used. However, when exogenous
[
H]PC was used as substrate in the presence of
PIP
, PLD stimulation by GTP
S was evident in the
membranes in the absence of cytosol or ARF. As will be discussed below,
these results can be explained by the presence of two PLD isoforms, one
responsive to ARF and another to Rho.
S-dependent PLD activity could also be detected in bovine brain
or rat liver cytosol.
(
)
This was barely
measurable in crude cytosol but came enriched after DEAE
chromatography.
S-dependent and showed very similar dependence on the
nucleotide. However, the basal activity of the membrane PLD was
significant, whereas that of the cytosolic PLD was barely detectable.
Furthermore, Ca
ions had very different effects on
cytosolic and membrane PLD. These data raise the possibility of
different PLD isozymes in the two subcellular locations, although this
remains speculative until the cytosolic and membrane enzymes are
purified and characterized.
S stimulation
of the membrane PLD could be inhibited by Rho-specific GDI
(27) . We observed similar results and also found that the
inhibition by Rho-GDI was more marked in the absence of cytosol
(Fig. 7 B). Furthermore, we observed that in membranes
extracted with Rho-specific GDI, it was possible to partially restore
the GTP
S stimulation by reconstitution with recombinant RhoA,
CDC42, and Rac1. Bovine sARF also restored PLD activity in the
extracted membranes, probably because of the presence of both
Rho-specific and ARF-specific PLD isozymes. Surprisingly, the
stimulation by Rho family G proteins did not show much specificity,
since all tested Rho recombinant proteins (RhoA, CDC42, Rac1) could
restore PLD activity in GDI-extracted or untreated membranes, although
to varying extents.
(
)
However, Western blotting
revealed that, of these proteins, only RhoA was detectable in the
membranes, implying that this was the Rho family member that exerted
endogenous control on the enzyme.
S (Fig. 10).
Among RhoA, RhoB, and CDC42, only RhoA was translocated in the presence
of nucleotide in a time-dependent manner. Again, Rac1 could not be
detected in either cytosol or membranes. These findings support the
involvement of RhoA in the regulation of membrane PLD.
(
)
Contrary to membrane PLD, cytosolic PLD responds to ARF,
but not to Rho, i.e. it must be different from one of the
membrane isozymes. The existence of different PLD isozymes makes it
possible to explain the different results obtained with membranes using
exogenous versus endogenous substrate. For example, although
an ARF-responsive isozyme is present in the membranes
(25, 26) , it cannot be responsible for the effects of
GTP
S added alone since ARF is lacking in the membranes. On the
other hand, a RhoA-responsive enzyme and RhoA are both present in the
membranes ( cf. Fig. 1 A and Fig. 10), and
this isozyme could therefore mediate the effects of GTP
S. If this
isozyme were able to utilize exogenous PC (under the specific assay
conditions) efficiently, this could explain the results. The existence
of other isozymes in the membranes that utilize PC less efficiently
could explain why a GTP
S effect is not detectable in the membranes
in the absence of cytosol or ARF when the endogenous substrate is
labeled.
S, guanosine 5`- 0-(3-thiotriphosphate); PC,
phosphatidylcholine; PE, phosphatidyl ethanolamine; PIP
,
phosphatidylinositol 4,5-bisphosphate; PEth, phosphatidylethanol; HAP,
hydroxylapatite; ARF, ADP ribosylation factor; sARF, soluble ARF;
Rho-GDI, Rho-specific GDP dissociation inhibitor; GST, glutathione
S-transferase; PA, phosphatidic acid.
S stimulation of PLD in
the GDI-extracted membranes but could significantly enhance the
stimulation when added to untreated membranes. This indicates that the
GDI treatment procedure produced additional changes that diminished the
ability of PLD to respond to these SMGs. Comparison of the figures also
shows that the relative potency of RhoA, Rac1, and CDC42 was changed.
We do not know the reason for these differences, but they could be due
to the extraction of GDP dissociation stimulator(s) and/or other
proteins that control the activity of these SMGs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.