(Received for publication, August 28, 1995; and in revised form, November 10, 1995)
From the
In HL60 cells, the membrane-bound phospholipase D (PLD) was
stimulated by 4-phorbol 12-myristate 13-acetate (PMA) in the
presence of the cytosolic fraction from HL60 cells or rat brain. The
cytosolic factor for this PMA-induced PLD activation was subjected to
purification from rat brain by sequential chromatographies. The PLD
stimulating activity was found in protein kinase C (PKC) fraction
containing
,
I,
II, and
isozymes. PKC isozymes
were further separated by hydroxylapatite chromatography. PKC
and
-
, but not
, isozymes were found to activate membrane-bound
PLD. PKC
was much more effective than PKC
for PLD activation.
Millimolar concentrations of MgATP were required for the PKC-mediated
PLD activation in HL60 membranes. MgATP is utilized to maintain the
levels of phosphatidylinositol 4,5-bisphosphate (PIP
) under
these assay conditions. The PKC-mediated PLD activation was completely
inhibited by neomycin, a high affinity ligand for PIP
, and
this suppression was recovered by the addition of exogenous
PIP
. Thus, these results suggest that PIP
is
supposed to play a key role in PKC-mediated PLD activity in HL60
membranes. Furthermore, PKC
-mediated PLD activation was
potentiated by the addition of recombinant RhoA protein in the presence
of guanosine 5`-O-(3-thiotriphosphate) (GTP
S). The
results obtained here indicate that PKC
and RhoA (GTP form) exert
a synergistic action in the membrane-bound PLD activation in HL60
cells.
Phospholipase D (PLD) ()has been recognized to play
an important role in signal transduction of many types of cells. PLD
hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid (PA)
and choline(1) . PA and its dephosphorylated product
diacylglycerol are important second messengers. PLD is activated in a
variety cells in response to receptor agonists, phorbol ester and
Ca
ionophore(2) . Recently, it has been
pointed out that several factors are required for activation of PLD. In
reconstitution experiments, activation of membrane-bound PLD induced by
phorbol 12-myristate 13-acetate (PMA) or nonhydrolyzable guanine
nucleotide (GTP
S) was observed only when cytosol and membranes
were present(3) . Similar findings were obtained in
permeabilized cell preparations in which leakage of cytosolic
components resulted in reduction of PLD
activity(4, 5) . These results imply that cytosolic
factors for PLD activation are involved in protein kinase C (PKC) and
GTP-binding proteins.
PKC has been reported to be implicated in PLD
activation in various cell types. Evidence that PKC up-regulates PLD
activity is supported by the observations that PKC inhibitors or PKC
down-regulation by long-term exposure to PMA prevents the increase of
PLD activity(2) . Recently, a role for specific PKC isozymes in
the regulation of PLD is presented by the studies overexpressing
PKC or -
isozymes in cells. Overexpression of PKC
I
enhances PMA-induced PLD activity in rat fibroblasts(6) .
Overexpression of PKC
in Swiss-3T3 fibroblasts (7) does
not induce an acute stimulation of PLD by PMA, but rather it plays a
role in the expression of PLD enzyme. Furthermore, in membranes
isolated from CCL39 fibroblasts(8) , only PKC
and -
are capable of activating PLD. However, exact molecular interactions
between PLD and PKC have not yet been studied.
On the other hand,
stimulation of PLD activity by GTPS in permeabilized cells and
cell lysates indicates its regulation by GTP-binding
proteins(2) . Recent studies have demonstrated the implication
of two small GTP-binding proteins, ADP-ribosylation factor (ARF) (9, 10) and Rho (11, 12) in the
regulation of PLD activity in several types of cells. Furthermore, in
some types of cells (13, 14, 15, 16, 17, 18, 19, 20) ,
PLD activation induced by both GTP
S and PMA was greatly enhanced,
compared with that caused by either stimulant alone. These findings
suggest that PKC may play an important role in positively modulating
GTP-binding protein-mediated PLD activity. However, the precise
relationship between GTP-binding proteins and PKC has not yet been
disclosed. Our recent study (20) has demonstrated that the
partially purified PKC fraction from rat brain cytosol showed a
synergistic stimulation of PLD activity of HL60 membranes by PMA and
GTP
S. Moreover, this synergistic activation of the membrane-bound
PLD was prevented by pretreatment with Rho GDP dissociation inhibitor
(RhoGDI), suggesting a potential role of RhoA in the PKC-mediated PLD
activation.
The present study was designed to gain more insight into
the mechanisms underlying the PMA-induced PLD activation in HL60
membranes. A cytosolic factor reconstituting PMA-induced PLD activity
was resolved as PKC fraction from rat brain. Among PKC, -
,
and -
isozymes, PKC
was the most effective in activating
membrane-bound PLD. PKC
-mediated PLD activation was
synergistically stimulated by RhoA in the presence of GTP
S.
Furthermore, MgATP and phosphatidylinositol 4,5-bisphosphate
(PIP
) were necessary for the membrane-bound PLD activation
by the partially purified PKC fraction which was free from small
GTP-binding proteins of brain cytosol.
Figure 1:
Activation of PLD in HL60 membranes by
cytosolic fractions. [H]Oleic acid-labeled HL60
membranes (50 µg of protein) and cytosolic fractions (50 µg of
protein) from HL60 cells or rat brain were incubated with 100 nM PMA, 10 µM GTP
S, or both PMA and GTP
S at 37
°C for 15 min in the presence of 0.3% butanol. PLD activity was
determined to measure the formation of [
H]PBut as
described under ``Experimental Procedures.'' Data represent
the mean ± S.D. of two different experiments, each carried out
in duplicate.
The
formation of phosphatidylalcohol such as PBut is commonly utilized to
monitor PLD activity(1, 2) . Although
[H] PBut has been reported to be metabolically
stable, its degradation in various assay conditions is not clearly
known. We have then analyzed the degradation of
[
H]PBut during the course of experiments.
[
H]PBut was prepared from PMA-stimulated intact
HL60 cells. The level of [
H]PBut decreased only
very little (less than 10%; statistically not significant) in the
reaction buffer during incubation (15 min). Additionally, membrane
fraction, cytosolic fraction, and stimulators (PMA and GTP
S) were
almost without effect (statistically not significant) on PBut
degradation in our incubation condition. Therefore, we examined PLD
activity by the formation of [
H]PBut.
Figure 2:
Activation of PLD in HL60 membranes by the
partially purified PKC fraction. [H]Oleic
acid-labeled HL60 membranes (50 µg of protein) and the indicated
concentrations of rat brain cytosol (A), the Mono Q-PKC
fraction (B), or the Superose-PKC fraction (C) were
incubated with 100 nM PMA or 10 µM GTP
S at
37 °C for 15 min in the presence of 0.3% butanol. PLD activity was
determined to measure the formation of [
H]PBut as
described under ``Experimental Procedures.'' Data represent
the mean of two different experiments.
Figure 3:
Mono Q anion exchange chromatography of
rat brain cytosol. Rat brain cytosol was subjected to Mono Q anion
exchange chromatography, and each fraction was assayed for its ability
to reconstitute HL60 membrane PLD activity and PKC activity as
described under ``Experimental Procedures.'' A,
[H]oleic acid-labeled HL60 membranes (50 µg
of protein) and 10 µl of each fraction were incubated with 100
nM PMA, 10 µM GTP
S, or both PMA and
GTP
S at 37 °C for 15 min in the presence of 0.3% butanol.
Western blot analysis of RhoA is shown in the inset. B, PKC
activity of each fraction was measured using MBP as a substrate in the
presence of 2 mM CaCl
, 10 nM PMA, and 50
µg/ml phosphatidylserine.
In order to separate
PKCs from small GTP-binding proteins, the Mono Q-PKC fraction was
subjected to Superose 12 gel filtration chromatography. The PKC
fraction eluted at a position of about 80 kDa was separated from the
fraction containing small GTP-binding proteins (less than 80 kDa) and
concentrated. This PKC fraction (Superose-PKC) contained ,
I,
II, and
isozymes, but Rho family small GTP-binding proteins,
RhoA, Rac1/Rac2, and Cdc42Hs were undetectable by Western blot analysis
(data not shown). The Superose-PKC fraction stimulated PLD activity in
response to PMA but failed to fulfill the stimulatory effect of
GTP
S (Fig. 2C). PLD activity was not detectable in
this PKC fraction using the
PE/PIP
/[
H]DPPC (16:1.4:1) substrate
system (data not shown) as described by Brown et
al.(9) , although crude rat brain cytosol contained weak
PLD activity as described above.
Figure 4:
Activation of PLD in HL60 membranes by the
Superose-PKC fraction and the translocation of PKCs to membranes. A, [H]oleic acid-labeled HL60 membranes
(50 µg of protein) and the Superose-PKC fraction (7.5 units/assay)
were incubated with the indicated concentrations of PMA or 4
-PDD
at 37 °C for 15 min in the presence of 0.3% butanol. B,
[
H]oleic acid-labeled HL60 membranes (50 µg
of protein) and the Superose-PKC fraction (7.5 units/assay) were
incubated with 100 nM PMA at 37 °C for the indicated times
in the presence of 0.3% butanol. PLD activity was determined to measure
the formation of [
H]PBut as described under
``Experimental Procedures.'' Data represent the mean of two
different experiments. C, the HL60 membranes (50 µg of
protein) and the Superose-PKC fraction (7.5 units/assay) were incubated
with 100 nM PMA at 37 °C for the indicated times. Proteins
in the HL60 membrane were electrophoresed and transferred to
nitrocellulose membrane. Western blot analysis was performed as
described under ``Experimental Procedures.'' D,
quantification of relative amounts of PKCs was performed by scanning
the spots on the film with a densitometer (Densitograph,
ATTO).
Figure 5:
Effect of MgATP on the PKC-mediated PLD
activation in HL60 membranes. A,
[H]oleic acid-labeled HL60 membranes were washed
once in MgATP-free buffer A. The washed membranes (50 µg of
protein) and the Superose-PKC fraction (7.5 units/assay) were incubated
with 100 nM PMA at 37 °C for 15 min in the presence of the
indicated concentrations of MgATP. PLD activity was determined to
measure the formation of [
H]PBut as described
under ``Experimental Procedures.'' Data represent the mean
± S.D. of two different experiments, each carried out in
duplicate. B, myo-[
H]inositol-labeled HL60 membranes
(50 µg of protein) were incubated with or without 0.5 mM MgATP at 37 °C for 15 min. [
H]PIP
was extracted as described under ``Experimental
Procedures.'' Data represent the mean ± S.D. of three
different experiments, each carried out in
duplicate.
In order to
further assess the involvement of PIP in PMA-mediated PLD
activation, we examined the effect of neomycin, which binds
polyphosphoinositides with high affinity. However, neomycin is reported
to inhibit PKC activity at higher concentrations (more than 2
mM)(38) . Therefore, the HL60 membrane fraction was
treated with neomycin (less than 1 mM) and then excess
neomycin was washed out prior to stimulation of partially purified PKC
fraction and PMA. The PKC-mediated PLD activity in HL60 membranes was
suppressed in parallel to increasing concentrations of neomycin (Fig. 6). 1 mM neomycin caused a complete inhibition
(approximately 95%) of the PLD activity. However, the suppressed
PKC-mediated PLD activity by 1 mM neomycin was restored by
addition of PIP
in a concentration-dependent manner (Fig. 6). In these experiments, PIP
was mixed with
phosphatidylethanolamine (PIP
/PE, 1:5 mol/mol), because of
effective incorporation into membranes. PE alone had no effect on
restoring PLD activity in neomycin-treated membranes (data not shown).
Figure 6:
Effects of neomycin and PIP on
the PKC-mediated PLD activation in HL60 membranes.
[
H]Oleic acid-labeled HL60 membranes were
incubated with the indicated concentrations of neomycin at 37 °C
for 10 min and then washed once in KCl- and NaCl-free buffer A. The
washed membranes were incubated with or without PIP
/PE
(1:5, mol/mol) liposomes containing indicated concentrations of
PIP
on ice for 30 min and then stimulated with 100 nM PMA at 37 °C for 15 min in the presence of the Superose-PKC
fraction (7.5 units/assay). PLD activity was determined to measure the
formation of [
H] PBut as described under
``Experimental Procedures.'' The responses were expressed as
percentages of the result obtained in the absence of neomycin and
exogenous PIP
. Data represent the mean ± S.D. of two
different experiments, each carried out in
duplicate.
Figure 7:
Hydroxylapatite chromatography of the
Superose-PKC fraction. The Superose-PKC fraction was applied to
hydroxylapatite column, and proteins were eluted with linear gradient
of potassium phosphate (20-300 mM). A,
[H]oleic acid-labeled HL60 membranes (50 µg
of protein) and aliquots (15 µl) of each fraction were stimulated
with 100 nM PMA, 10 µM GTP
S, or both PMA and
GTP
S at 37 °C for 15 min in the presence of 0.3% butanol. PLD
activity was determined to measure the formation of
[
H]PBut as described under ``Experimental
Procedures.'' B, proteins from each fraction were
subjected to Western blot analysis with PKC isozyme-specific
antibodies.
The effects of these purified
PKC isozymes on HL60 membrane PLD activity were examined. In the
presence of PMA (100 nM), PLD activity was enhanced by
PKC or -
in a concentration-dependent manner (Fig. 8, A and B). PKC
was the most effective with a
maximal effect obtained at about 10 units/assay. The maximal PLD
activation obtained at around 10 units/assay of PKC
was almost
half of that induced by PKC
. PKC
had no effect on PLD
activation in HL60 membranes (data not shown). The PLD activity
stimulated by PKC
(2.5 units/assay) plus
(2.5 units/assay)
was the same as that obtained with PKC
alone (Fig. 8C). GTP
S in the absence of PMA did not
stimulate PLD activity. However, in the presence of both PMA and
GTP
S, the PKC-mediated PLD activity was synergistically
potentiated (Fig. 8). Additionally, recombinant PKC
stimulated the membrane-bound PLD activity in HL60 cells in a quite
similar manner as observed with the purified PKC
fraction (data
not shown).
Figure 8:
Activation of PLD in HL60 membranes by
PKC and
. [
H]Oleic acid-labeled HL60
membranes (50 µg of protein) were stimulated with 100 nM PMA, 10 µM GTP
S, or both PMA and GTP
S at 37
°C for 15 min in the presence of the indicated concentrations of
the PKC
fraction (A) and PKC
fraction (B). C, [
H]oleic acid-labeled HL60 membranes
(50 µg of protein) were stimulated with 100 nM PMA or both
PMA and 10 µM GTP
S at 37 °C for 15 min in the
presence of PKC
(2.5 units/assay) and/or PKC
(2.5
units/assay). PLD activity was determined to measure the formation of
[
H]PBut as described under ``Experimental
Procedures.'' Data represent the mean ± S.D. of two
different experiments, each carried out in
duplicate.
Figure 9:
Effect of RhoA on PLD activation in HL60
membranes. A, [H]oleic acid-labeled HL60
membranes (50 µg of protein) were stimulated with the indicated
concentrations of recombinant RhoA at 37 °C for 15 min in the
presence or absence of 10 µM GTP
S. B,
[
H]oleic acid-labeled HL60 membranes (50 µg
of protein) were stimulated with 100 nM PMA, 10 µM GTP
S, or both PMA and GTP
S at 37 °C for 15 min in
the presence of PKC
(5 units) and/or RhoA (20 nM). PLD
activity was determined to measure the formation of
[
H]PBut as described under ``Experimental
Procedures.'' Data represent the mean ± S.D. of two
different experiments, each carried out in
duplicate.
Figure 10:
Translocation of PKC and RhoA to
membranes in HL60 cells. The HL60 membranes (50 µg of protein) and
HL60 cytosol (50 µg of protein) were stimulated with 100 nM PMA, 10 µM GTP
S, or both PMA and GTP
S at 37
°C for 15 min. Proteins in the HL60 membrane were electrophoresed
and transferred to the nitrocellulose membrane. Western blot analysis
with anti-PKC
antibody (A) and anti-RhoA antibody (B) was performed as described under ``Experimental
Procedures.''
Several factors have been implicated in the regulation of PLD
activity, such as Ca, PKC, protein-tyrosine kinase,
and GTP-binding proteins(2) . However, their detailed
mechanisms are not fully understood. Recently, PLD assay systems using
permeabilized cells or cell-free preparations have been developed, and
cytosolic factors including ARF and Rho protein are identified as
regulatory factors for PLD activity. PMA, know as a PKC activator,
activates PLD in many types of cells. Although the effect of PMA is
assumed to be mediated through PKC, the pathway leading to PLD
activation remains to be disclosed. In the present study, the mechanism
of PMA-induced PLD activation was investigated in HL60 membranes.
In
the cell-free system using isolated membranes from HL60 cells, the
membrane-bound PLD activity was stimulated by PMA in the presence of
the cytosolic fractions from HL60 cells or rat brain. The cytosolic
factors required for this activity were partially purified by
sequential chromatographies and identified as PKCs. Our previous report (20) has shown that the PKC-mediated PLD activation was most
effectively induced in the presence of Ca. In
addition, PKC
, -
I, -
II, and lesser amounts of -
isozyme were observed, but PKC
, -
, -
, -
, and
-
isozymes were undetectable in HL60 cells by Western blot
analysis (data not shown). These results suggest that conventional PKC
(cPKC) isozymes may play a role in PMA-stimulated PLD activation in
HL60 membranes. We have further examined PKC isozymes involved in the
regulation of membrane-bound PLD. The results indicate that PKC
and -
activate the membrane-bound PLD and also that PKC
is
much more effective in its activation in HL60 cells. The study of the
regulation of PLD in membranes isolated from Chinese hamster lung
(CCL39) fibroblasts demonstrated that addition of purified PKC
and
-
from rat brain could activate PLD(8) . In our study,
PKC
and -
did not additively activate the PLD in HL60
membranes (Fig. 8C), suggesting that both PKC isozymes
act at the same step for the PLD activation.
Previously, Tettenborn and Mueller (40) demonstrated that the PLD activation by PMA was dependent on the presence of ATP in HL60 cell lysates. Olson et al. (3) also reported that PMA-induced PLD activation was dependent on ATP in the neutrophil cell-free system. The PKC-mediated PLD activation in HL60 membranes required MgATP at millimolar concentrations (Fig. 5). Our previous study (20) has shown that the PKC-mediated PLD activation in HL60 membranes was not suppressed by Ro31-8425, a potent PKC inhibitor. Similar findings were reported by Conricode et al. (41) showing that membrane-bound PLD in CCL39 fibroblasts could be activated by PKC in a phosphorylation-independent mechanism and that the PLD activation by PKC was observed even in the absence of ATP, suggesting that PKC may activate PLD by an allosteric mechanism without ATP-dependent phosphorylation. On the other hand, it was reported more recently that the effect of ATP on PKC-mediated PLD activation is mediated by phosphorylation in human neutrophils(42) . Although this discrepancy could reflect difference in cell type, our present data cannot exclude this possibility.
Several recent studies have
provided evidence that PIP may act as an important cofactor
for PLD activity. Brown et al. (9) developed a
reconstitution system for solubilized PLD activity from HL60 cells in
which the substrate PC was present in the form of mixed phospholipid
micelles including PIP
and demonstrated the requirement of
PIP
in the ARF-mediated PLD activation. Liscovitch et
al. (35) have shown that the activity of partially
purified PLD from brain membranes was stimulated considerably by
PIP
. In permeabilized U937 cells(37) , GTP
S
was observed to elevate the levels of polyphosphoinositides in the
presence of MgATP, and either GTP
S- or PMA-induced PLD activation
was prevented by the antibody against phosphatidylinositol 4-kinase.
Furthermore, neomycin, a high affinity ligand for PIP
,
inhibited the activity of purified PLD from brain membranes (35) and GTP
S-induced PLD activation in permeabilized HL60
cells(15) , human neutrophils(17) , and U937
cells(37) . The results obtained in the present study also
indicated that PIP
was required for PKC-mediated PLD
activation in HL60 membranes. In fact, the level of PIP
in
HL60 membranes was increased by incubation with MgATP (Fig. 5B). Therefore, MgATP is supposed to play a key
role in maintaining the level of PIP
for PLD activity. The
effect of PIP
might be partly explained by the fact that
PIP
(100 µM) caused little enhancement
(approximately 1.4-fold) of PKC activity (data not shown). However, at
present, detailed information for the site of action of PIP
and involvement of protein phosphorylation is not available and
should be obtained by additional experiments which are currently in
progress in our laboratory.
The PKC-mediated PLD activation in HL60
membranes was potentiated by the addition of GTPS (Fig. 8).
This finding led us to assume that translocated PKC
or -
interacts with membrane-associated GTP-binding proteins, resulting in a
synergistic activation of PLD. Several lines of evidence are present to
indicate that activation of PLD is mediated by small GTP-binding
proteins which interact directly with the solubilized
PLD(9, 39, 43, 44, 45) . In
addition, it was demonstrated that PIP
synthesis is
regulated by Rho family small GTP-binding
proteins(46, 47) . Our previous study (20) has
shown that RhoGDI, Rho GDP dissociation inhibitor which extracts Rho
proteins from membranes, prevented the synergistic effect by GTP
S
in PKC-mediated PLD activation in HL60 membranes, and also that this
suppressed PLD activation was restored by the addition of recombinant
RhoA. These results suggest the interaction between PKC and
membrane-associated RhoA. Western blot analysis with the antibody to
RhoA showed the presence of RhoA in the HL60 membrane fraction (data
not shown). Furthermore, the present study showed that
PKC
-mediated PLD activity was potentiated by the addition of
recombinant RhoA in the presence of GTP
S (Fig. 9). It was
also shown that cytosolic PKC
and RhoA were translocated to
membranes in a PMA- and GTP
S-dependent manner, respectively. Most
recently, Siddiqi et al. (39) indicated that not only
RhoA but also Rac and Cdc42Hs induced PLD activation in HL60 membranes.
Among the Rho family GTP-binding proteins, however, only RhoA was
translocated to HL60 membranes in the presence of
GTP
S(39) . These findings suggest the involvement of RhoA
in the regulation of membrane-bound PLD in HL60 cells.
Despite many
studies, the exact mechanism for the implication of PKC in PLD
activation has not yet been defined. It may be possible that PKC
interacts directly with PLD in membranes or that PKC interacts with
other membrane-associated proteins that in turn activates PLD. In the
present study, the PKC fraction free from small GTP-binding
proteins was observed to activate the membrane-bound PLD (Fig. 8). However, GTP
S synergistically enhanced
PKC-mediated PLD activation. This synergistic activation by GTP
S
was prevented by pretreatment of HL60 membranes with RhoGDI, as shown
in the previous study(20) . Recent reports demonstrate that
ARF-mediated PLD activation is modulated by other as yet unidentified
cytosolic proteins(48, 49, 50) . RhoA may act
as a PKC-modulating factor in PKC-mediated PLD activation. We have
recently obtained a preliminary finding that in the membranes
pretreated with GDP
S, the PKC
-mediated PLD activation by PMA
was almost completely abolished. Thus, the PKC-mediated PLD activation
mechanism involving RhoA appears to be more complex than expected. The
reconstitution system using purified membrane-bound PLD will add
further insight into the mode of the synergistic activation of
membrane-bound PLD in HL60 cells.