(Received for publication, January 12, 1995; and in revised form, June 16, 1995)
From the
In a variety of intact cells, phorbol esters are known to
activate phospholipase D. In a cell-free system consisting of plasma
membrane and cytosol from human neutrophils, phorbol esters activated
phospholipase D in an adenosine nucleotide triphosphate-dependent
manner. ATPS (adenosine 5`-O-(thiotriphosphate)) was
2-3-fold more effective than ATP, while ADP and AppNHp
(adenyl-5`-yl imidodiphosphate) were ineffective, and activation was
blocked by the kinase inhibitor staurosporine. In cytosol depleted of
protein kinase C by chromatography on threonine-Sepharose, phorbol
ester-dependent activation was lost, but was restored upon addition of
purified rat brain protein kinase C. The target for phosphorylation was
shown to be the plasma membrane: plasma membrane was phosphorylated
using ATP
S/phorbol 12,13-dibutyrate and protein kinase C and was
reisolated to remove activators. Upon adding nucleotide-depleted
cytosol, activator-independent phospholipase D activity was
seen. Using this prephosphorylation protocol, PKC-dependent activation
of plasma membranes was found to require micromolar calcium,
implicating a conventional protein kinase C. Using recombinant isoforms
of protein kinase C, only the conventional isoforms showed significant
activation, with the following rank order of potency:
>
>
; the
,
,
,
, and
isoforms showed little or no activity. Thus,
conventional isoform(s) of protein kinase C activate neutrophil
phospholipase D by phosphorylating a target protein located in the
plasma membrane.
Phospholipase D (PLD) ()catalyzes the hydrolysis of
phospholipids to generate phosphatidic acid plus the headgroup. In
eukaryotic cells the enzyme is relatively specific for
phosphatidylcholine. Phosphatidic acid may act directly as a signal
molecule (1, 2) or can be metabolized to form
diacylglycerol (DG) by phosphatidic acid phosphohydrolase. The latter
can function as an activator of protein kinases C (PKC) and possibly
other diacylglycerol-dependent enzymes. In the presence of primary
alcohols, PLD also catalyzes a unique transphosphatidylation reaction
to produce the phosphatidylalcohol (e.g. phosphatidylethanol),
which can be used as a convenient measure of PLD activity(3) .
PLD can be activated by a variety of agonists, including those that act
via some G protein-coupled receptors and via receptor tyrosine kinases (2, 4, 5) (reviewed in (2) ). In a
wide variety of cells, phorbol esters are also potent activators of
PLD. In human peripheral blood neutrophils, both fMet-Leu-Phe, which
acts via a G protein-coupled receptor, and phorbol esters, which
presumably act through PKC, activate
PLD(6, 7, 8) .
Although receptor-coupled
PLD enzyme(s) have not been purified, cell-free systems have advanced
the understanding of the enzymology and regulation of PLD. Exton and
colleagues (4) observed GTPS-activated PLD in liver plasma
membranes. Cell-free reconstitution of PLD activity in neutrophil
fractions requires the participation of protein components not only in
the plasma membrane, but also in the cytosol(7, 9) .
Activation was achieved using either GTP
S or phorbol esters, and
required micromolar calcium(7) . In the neutrophil system, one
of the plasma membrane components is a Rho family small molecular
weight GTP-binding protein(10) . RhoA has been described in
liver plasma membranes as the PLD-activating GTP-binding
component(11) . Another small GTP-binding protein, ARF, was
purified from brain cytosol as a factor which activates the PLD in
HL-60 membranes(12, 13) . Under our conditions, ARF is
not essential for PLD activation, but synergizes with an essential
50-kDa cytosolic factor(14) , the identity of which is not yet
clear.
PKC plays an important role in signal transduction,
regulating a variety of cellular functions via phosphorylation of
target proteins. PKC is comprised of a family of related enzymes that
are differentially expressed in a variety of tissues and cell
types(15, 16, 17) . To date, 10 PKC
isoenzymes have been identified and can be categorized into three
groups (conventional, novel, and atypical) based on functional
properties as well as sequence. The conventional PKCs (cPKC,
,
, and
) require calcium and
phospholipid, and are activated by DG or phorbol ester. The novel PKCs
(
,
,
, and
) are also activated by diacylglycerol
or phorbol esters and require phospholipid, but do not require calcium.
On the other hand, the atypical PKCs (
and
) are not
activated by DG or phorbol esters but are dependent on
phosphatidylserine.
Activation of PLD by phorbol esters is assumed
to occur via one or more PKCs, but other effects of phorbol esters not
involving PKC have been suggested. In addition, in liver plasma
membranes, PLD activation by phorbol esters, while requiring PKC, is
independent of ATP, suggesting a phosphorylation-independent mechanism (18) (e.g. direct complexation of PKC with PLD). In
the cell-free system from neutrophil, phorbol ester-dependent
activation of PLD requires ATP and is inhibited by
staurosporine(7) , suggesting a classical
phosphorylation-dependent mechanism. In the present studies, we
utilized biochemical approaches to investigate the function of PKC in
the activation of neutrophil PLD. We find that activation requires
conventional isoforms of PKC (,
, or
) and
that the target of phosphorylation is in the plasma membrane.
Figure 1:
Adenine nucleotide
specificity for PMA stimulation of phospholipase D. Isolated plasma
membranes (25 µg) and nucleotide-depleted cytosol (50 µg) were
incubated in buffer containing 1 µM CaCl for
20 min at 37 °C with either 10 µM GTP
S or with
100 nM PMA in the presence or absence of 1 mM each of
the following adenine nucleotides: ADP, ATP, ATP
S, or AppNHp as
detailed under ``Experimental Procedures.''
[
H]Phosphatidylethanol was quantified as
described under ``Experimental Procedures'' and is expressed
as the percentage of total counts in lipids. Basal PEth in the absence
of activators or nucleotides was 1 ± 0.2%, and this value was
subtracted from the values shown. Each value is the mean ± S.E.
of three to five experiments, each using a separate preparation of
plasma membrane and cytosol performed in
duplicate.
The dose dependence for adenosine nucleotides in PMA-activated PLD
is shown in Fig. 2. PMA in the absence of nucleotides failed to
activate. ATP showed a broad concentration optimum (10 µM to 1 mM), while that for ATPS was 1 mM. The
maximal response with ATP, however, was only about one third of the
maximal response seen with ATP
S. The latter response in this
experiment was similar to that seen with GTP
S (filledtriangle, lowerpanel). Staurosporine
(1 µM), a protein kinase inhibitor, almost completely
inhibited PMA stimulated PLD activity at all concentration ranges of
both ATP and ATP
S. Thus, these data are consistent with both ATP
and ATP
S serving as phosphate donors, activating PLD in a
PKC-dependent phosphorylation reaction. In addition, when
[
S]ATP
S was used, a large number of
proteins in the plasma membrane were phosphorylated in a PMA-dependent
manner, indicating facile transfer of the thiophosphoryl group via PKC
(data not shown).
Figure 2:
Concentration dependence of ATP or
ATPS for PMA stimulation of PLD and inhibition by staurosporine.
Isolated plasma membranes (25 µg) and nucleotide-depleted cytosol
(50 µg) were incubated in buffer containing 1 µM
CaCl
with 10 either µM GTP
S (filledtriangle, lowerpanel) or with 100
nM PMA plus the indicated concentration of either ATP (upperpanel, filledcircles) or
ATP
S (lowerpanel, filledcircles) as detailed under ``Experimental
Procedures.'' ATP or ATP
S alone were also varied in the upper and lowerpanels, respectively, and
PLD activity measured (opencircles). Staurosporine
(1 µM) was included along with ATP or ATP
S in the upper and lowerpanels (opentriangles), respectively. Reactions were incubated for 20
min at 37 °C and terminated, and PEth was quantified as described
under ``Experimental Procedures.'' Basal PEth formed in the
absence of activators and inhibitors was 0.5 ± 0.2% (diamond, lowerpanel). Each value
represents the mean ± range of two experiments each using
separate preparations of cell fractions, with each point in each
experiment the average of duplicate
determinations.
The likely explanation for the enhanced
effectiveness of ATPS compared with ATP is that the thiophosphoryl
group, once transferred to the protein, is resistant to the action of
phosphatases, thereby increasing the stability of the phosphorylated
form of the protein. Dubyak and colleagues (27) showed that in
a cell-free system from U937 cells, inhibitors of protein phosphatases
such as vanadate augmented the activation of PLD. As shown in Fig. 3, under our conditions, neither vanadate nor okadaic acid
augmented PMA-stimulated activity when either ATP or ATP
S were
used. Dose dependence curves for these compounds were also carried out
(not shown), and enhancement of activity with ATP was not seen at any
concentration used. Thus, phosphatase inhibitors in this system did not
appear to be useful for preserving the activated state.
Figure 3:
Effects of okadaic acid and vanadate on
PMA-stimulated phospholipase D activity. Isolated plasma membranes (25
µg) and nucleotide-depleted cytosol (50 µg) were incubated in
buffer containing 1 µM CaCl as indicated
either with 10 µM GTP
S or with/without 100 nM PMA in the presence of 1 mM ATP or 1 mM ATP
S. Okadaic acid (10 µM) or vanadate (100
µM) were also included as indicated, and the reaction
mixture was incubated for 20 min at 37 °C. Reactions were
terminated and PEth quantified as described under ``Experimental
Procedures.'' PEth was 1.6 ± 0.5% in the absence of any
added activators or inhibitors, and this value was subtracted from
those shown. Each value represents the mean ± range of two
experiments, each carried out with a separate preparation of
subcellular fractions and done in
duplicate.
The increase
in [H]PEth seen with PMA/ATP
S was apparently
due exclusively to stimulated formation, and there was no effect on its
subsequent metabolism, which is generally assumed to be minimal. To
verify this assumption, we included 0, 40, and 400 nM unlabeled PEth in an incubation. This represents about 10- and
100-fold more PEth than would be formed during the incubation. If PEth
were unstable in the incubation mixture, then cold PEth should compete
for metabolism, thereby enhancing the recovery of the
[
H]PEth formed during the incubation. In fact,
unlabeled PEth added to the incubation had no effect (data not shown).
Figure 4:
Separation of PKC from neutrophil cytosol
by column chromatography. PanelA, neutrophil cytosol
(60 mg protein) was chromatographed at 4 °C using DEAE followed by
threonine-Sepharose column, as detailed under ``Experimental
Procedures.'' At the arrow (inset), buffer
containing 1 M NaCl was added. Fractions (4 ml) were
collected, and an aliquot of each was analyzed for PKC activity (inset), using histone phosphorylation as described under
``Experimental Procedures.'' Fractions were pooled as
indicated, concentrated, and assayed for PLD-supporting activity as
detailed under ``Experimental Procedures,'' using either 10
µM GTPS or 100 nM PMA plus 1 mM
ATP
S as activators in the presence of 1 µM
CaCl
. PanelB, samples were analyzed for
the indicated isoforms of protein kinase C, using immunoblotting with
isoform-specific antisera, as described under ``Experimental
Procedures.'' Lanes are as follows: lane1,
neutrophil cytosol (60 µg); lane2, pooled
fractions 1-22 (60 µg); lane3, pooled
fractions 27-30 (60 µg); lane4, 1 µg
each of purified recombinant protein kinase C
,
,
,
, and
, respectively (top to bottom); lane5, 1 µg each of purified recombinant protein
kinase C
,
,
,
,
, respectively
(top to bottom, used to demonstrate antibody
specificity).
To verify that all isoforms of PKC had been removed from the
``PKC-depleted cytosol,'' immunoblotting was carried out on
cytosol, on PKC-depleted cytosol (fractions 1-22) and on the
PKC-containing pooled material (fractions 27-30). No PKC isoforms
were detected in the PKC-depleted pool. However, ,
,
, and
isoforms were detected
in both the cytosol and in pooled fractions 27-30 (Fig. 4B). The
isoform was detected at a low
level in neutrophil cytosol (Fig. 4B), but was not seen
in fractions 27-30, indicating that this isoform remained
associated with the threonine-Sepharose column. PKC
,
, and
were not detected, consistent with other reports.
Fractions
1-22 were pooled, concentrated, and used to determine if PLD
activity could be reconstituted with purified rat brain PKC. As shown
in Table 1, PMA/ATPS-stimulated PLD activity was seen when
plasma membranes were incubated with native cytosol, but no activity
was seen when PKC-depleted cytosol was used. However, when plasma
membranes were incubated with PKC-depleted cytosol plus purified rat
brain PKC (a mixture of isoforms), PMA/ATP
S-stimulated PLD
activity was recovered to a level slightly higher than that seen with
native cytosol.
Membranes preincubated with PDBu/ATPS alone did not show
significant activity upon recombination with cytosol, indicating that
carryover of the activating factors into the incubation was not a
problem. Using radiolabeled [
S]ATP
S in the
incubation/reisolation protocol, 0.6% was carried over into the plasma
membrane fraction, and using [
H]PDBu, 3% was
recovered with plasma membranes. Thus, the final concentration of
ATP
S carried over into the second incubation was 6
µM, well below the concentration needed to support phorbol
ester-dependent activation (see Fig. 2). Although a potentially
activating concentration of PDBu (30 nM) may have been carried
through, this was not a problem since the ATP
S was effectively
removed. Also, preincubation with cytosol alone did not permit
activator-independent activation. As a positive control to demonstrate
that the PLD activity was still present after reisolation of plasma
membranes, these preparations were stimulated with PMA/ATP
S in the
presence of added cytosol (right-hand data column, Table 2).
Concentration dependence curves using highly
purified preparations of the major activating isoforms were also
carried out and are summarized in Fig. 5. All three of the
conventional isoforms were active, but there was a demonstrable rank
order of specificity for the three, with PKC >
PKC
> PKC
, which showed respective EC
values
of 0.6, 5, and 20 ng/ml, respectively. The maximal activation of PLD by
all effective PKC isoforms was similar. Several concentrations in this
same range of non-conventional and atypical isoforms of PKC were also
tested. None of these produced significant activation. A complete
concentration dependence curve was also carried out for PKC
(Fig. 5, lowerpanel), which showed no
activity at any concentration tested.
Figure 5:
Concentration dependence for activation of
phospholipase D by isoforms of protein kinase C. The concentration of
PKC was varied as indicated in the preincubation (5 min at 30 °C)
with plasma membrane in the presence of 1 µM phorbol
12,13-dibutyrate plus 1 mM ATPS and 5 µM CaCl
. Plasma membranes were then reisolated on a
discontinuous sucrose gradient, recombined, and incubated with
nucleotide-depleted cytosol as detailed under ``Experimental
Procedures.'' No further addition of activators was made to the
incubation. Each experiment was repeated two to three times with
duplicate incubations each time for each isoform of PKC; points shown
represent the average of all determinations. The isoform for each
titration is indicated in each panel.
The present study has investigated the role of PKC in the activation of neutrophil PLD. Activation of PLD by phorbol esters has been widely documented in a variety of tissues (for review, see (2) ), and it is assumed that PKC participates directly or indirectly in this process. In some cells, down-regulation of PKC elicited by chronic exposure to phorbol esters prevents activation of PLD by phorbol esters as well as by other agonists(28, 29, 30) . Nevertheless, PKC-independent mechanisms of phorbol ester activation have been suggested in some cases(31, 32, 33) . In one of the few studies carried out in a cell-free system, Conricode et al.(18) showed that phorbol ester activation in cell membranes from a lung fibroblast cell line, while dependent on PKC, was independent of ATP and protein phosphorylation. Inhibitors of PKC such as staurosporine and H-7, which act competitively at the ATP-binding site, are inhibitory toward PMA activation in some systems(7, 34, 35) , while failing to inhibit in others(29, 34) . Phosphorylation-independent activation suggests a direct complex formation between PKC and a PLD-related protein. In this regard, a protein ``receptor'' for PKC has been described recently (37) , and such a binding target might be related to PLD activation.
Studies in neutrophils,
however, support a classical picture of PKC action involving
phosphorylation of a target protein. Previous studies in our laboratory (7) and others (38) have demonstrated that PLD
activation by phorbol esters required ATP. Nevertheless, activation by
phorbol esters in the presence of ATP was consistently less effective
than that by GTPS. In the present studies, which utilize a
cell-free system, we confirm that phorbol ester activation requires ATP
or an ATP analog. In addition, we find that when ATP
S was used in
place of ATP, phorbol esters activated to an extent as great or greater
than GTP
S. Consistent with a phosphorylation mechanism,
staurosporine inhibited PMA activation when either ATP or ATP
S
were used but did not inhibit activation by GTP
S(7) . (
)This rules out the possibility that ATP
S might be
acting via a nucleoside diphosphate kinase to generate GTP
S. Thus,
the most likely mechanism for the enhanced effectiveness of ATP
S
over ATP is that the thiophosphoryl group, once transferred to the
protein target, is more stable to the effects of endogenous
phosphatases than is the phosphate group, as has been seen in other
studies. Some protein phosphatase inhibitors were also tested for
possible stimulatory effects when ATP was used. Vanadate is a tyrosine
phosphatase inhibitor, while okadaic acid inhibits protein phosphatase
1 and 2A. Both inhibitors have been shown previously to augment phorbol
ester-stimulated PLD activity in U937 cells and in intact and
permeabilized neutrophils(39, 40, 41) .
However, neither of these inhibitors enhanced the PMA-stimulated PLD
activity in our cell-free system. This may indicate that other
phosphatases play a role in this setting and/or that the effects seen
by other workers may be due to phosphorylations higher up in the
activation pathway.
Further support for a classical mechanism for
phorbol ester/PKC activation came from studies in which PKC was
depleted from the cytosol with loss of PMA-dependent activity (Fig. 4A and Table 1). Interestingly, activation
by GTPS was retained, indicating that the activation by the
GTP-binding protein does not require the direct participation of PKC.
Addition of rat brain PKC to the depleted system reconstituted activity (Table 1). Activity retained its absolute dependence on cytosol,
indicating that the cytosolic factor was not PKC itself. We have
recently found that the cytosolic factor is an approximately 50-kDa
protein, and that this factor synergizes with ADP-ribosylation factor
in the activation of PLD(14) .
Major isoforms of PKC that
are present in human neutrophils include conventional (,
, and
), novel (
and
),
and atypical (
) forms (42, 43, 44) , (
)and are indicated in Table 4. Our studies confirm
the presence of these isoforms in neutrophil cytosol. In addition, we
show that these isoforms are completely removed from cytosol by
threonine-Sepharose chromatography, thus enabling us to reconstitute
the system with individual PKC isoforms. The present studies implicate
conventional (calcium-dependent) but not other isoforms of PKC in the
activation of neutrophil PLD. Our preincubation/reisolation protocol
allowed us to separately evaluate the calcium requirement of the
PLD-activating PKC isoforms present in neutrophil cytosol and the
calcium dependence of the PLD itself. This was important, since both
PMA- and GTP
S-dependent activation require a submicromolar
concentration of calcium, which is presumably due to a calcium
requirement for the phospholipase itself(7) . Using this
two-stage protocol, we found that the activating PKC present in cytosol
was calcium-dependent, implicating a conventional isoform. This was
confirmed using purified recombinant PKC isoforms (Table 4). Only
the conventional isoforms resulted in significant activation. When dose
responses were carried out, the PLD activation showed the following
rank order of specificity for PKC: PKC
> PKC
> PKC
. Interestingly, the
isoform, which
differs from PKC
by 50 residues at the C terminus, was
completely inactive. Our studies are in agreement with studies in rat
fibroblasts in which PKC
overexpression was
accompanied by an increase in PMA-activated
PLD(45, 46) . Overexpression of PKC
in Swiss/3T3
fibroblasts also resulted in enhanced PLD activity, but this was
attributed to increased induction of the lipase itself(47) . In
membranes from a lung fibroblast cell line, Exton and colleagues (48) also found that only conventional forms of PKC activated,
but in this case, the effect of
was greater than
, and
showed no activity. Notably, in contrast to our cell-free system,
phorbol ester/PKC activation did not require protein phosphorylation
and occurred in the absence of cytosol(18) . Insel and
colleagues (49) used antisense technology to reduce levels of
either PKC
or
in Madin-Darby canine kidney cells.
Elimination of the
but not the
form resulted in a 70% loss
in PLD activity. In contrast, several in vivo studies (50, 51, 52) that were based on correlative
data or on selective down-regulation of PKC isoforms have implicated
non-conventional forms of PKC, in particular PKC
. Although the
neutrophil is not known to contain this isoform, we investigated its
function, both at a single concentration (Table 4) and in a dose
dependence curve (Fig. 5). No activity was seen at any
concentration used, indicating that the neutrophil PLD cannot be
functionally linked to this isoform. Finally, PLD activity has been
reported in one case to be inhibited through the action of
PKC(53) . Notably, in our studies, the PKC
(and, to a
lesser extent,
) showed inhibitory phases at higher levels of the
kinase. This may indicate that there are not only activating but also
inhibitory phosphorylations by some isoforms of PKC. Differences seen
in PKC isoform selectivity may reflect different isoforms of PLD that
are likely to be present in different tissues(2) .
Because
reconstitution of PLD activity in neutrophil requires both plasma
membrane and cytosol, it was important to define which fraction
contained the target for PKC phosphorylation. Using the two-stage
activity assay (i.e. prephosphorylation of the membranes
followed by reisolation and addition of cytosol), we have shown that
the major target of an activating phosphorylation is in the plasma
membrane. Notably, PKC, which showed the lowest EC
in
our system, is the major membrane translocating form of PKC in human
neutrophils(42, 43) , consistent with a plasma
membrane target. The particular protein target of phosphorylation
remains an area for future study. A fairly large number of proteins in
the plasma membrane became thiophosphorylated with
[
S]ATP
S, complicating the identification of
a particular target. Isozyme selectivity may be useful in this regard
for narrowing the list of candidates. To date, the only documented
PLD-related protein component in neutrophil plasma membranes is a Rho
family small GTPase(10) , possibly RhoA(11) . In this
regard, studies in several systems have pointed to synergy between
phorbol ester and guanine nucleotide
activation(27, 54) , and we have shown that inhibition
of Rho with RhoGDI or GDP inhibits phorbol ester activation, (
)consistent with a cooperative role for these two
activators. Although it is possible that Rho is a direct target of
phosphorylation, it seems likely that other components (e.g. the PLD itself) that are likely to be present in the plasma
membrane may be the actual target of phosphorylation. It is also
possible that the PKC activation induces other changes in the plasma
membrane such as changes in the phospholipid composition (e.g. phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol
1,4,5-trisphosphate), and that it is these changes rather than the
protein phosphorylation itself that cause activation of phospholipase
D. In this regard it is interesting to note recent studies of
phospholipase D in U937 cells, in which phosphatidylinositol
4,5-bisphosphate was implicated as a regulator of
activity(55) . It will be important in the future to isolate
and reconstitute the individual PLD-related components and to
investigate their regulation by phosphorylation.