(Received for publication, November 17, 1994)
From the
Phospholipase D (PLD) has been implicated in signal transduction
and membrane traffic. We have previously shown that
phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P)
stimulates in vitro partially purified brain membrane PLD
activity, defining a novel function of PtdIns-4,5-P
as a
PLD cofactor. In the present study we extend these observations to
permeabilized U937 cells. In these cells, the activation of PLD by
guanosine 5`-3-O-(thio)triphosphate (GTP
S) is greatly
potentiated by MgATP. We have utilized this experimental system to test
the hypothesis that MgATP potentiates PLD activation by G proteins
because it is required for PtdIns-4,5-P
synthesis by
phosphoinositide kinases. As expected, MgATP was absolutely required
for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P)
and PtdIns-4,5-P
levels in the permeabilized cells. In the
presence of MgATP, GTP
S further elevated the levels of the
phosphoinositides. The importance of PtdIns-4,5-P
for PLD
activation was examined by utilizing a specific inhibitory antibody
directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the
enzyme responsible for the first step in the synthesis of
PtdIns-4,5-P
. Anti-PtdIns 4-kinase completely inhibited
PtdIns 4-kinase activity in vitro and reduced by 75-80%
PtdIns-4-P and PtdIns-4,5-P
levels in the permeabilized
cells. In parallel, the anti-PtdIns 4-kinase fully inhibited the
activation of PLD by GTP
S and caused a 60% inhibition of PLD
activation by the phorbol ester
12-O-tetradecanoylphorbol-13-acetate, indicating that elevated
PtdIns-4,5-P
levels are required for PLD activation. This
conclusion is supported by the fact that neomycin, a high affinity
ligand of PtdIns-4,5-P
, also blocked PLD activation.
Furthermore, the activity of PLD in U937 cell lysate was stimulated by
PtdIns-4,5-P
in a dose-dependent manner. The current
results indicate that PtdIns-4,5-P
synthesis is required
for PLD activation in permeabilized U937 cells and strongly support the
proposed function of PtdIns-4,5-P
as a cofactor for PLD. In
addition, the results further establish PtdIns-4,5-P
as a
key component in the generation of second messengers via multiple
pathways including phosphoinositidephospholipase C, phosphoinositide
3-kinase and PLD.
Receptor-mediated hydrolysis of cellular phospholipids is a
ubiquitous biochemical event of central importance in cell signal
transduction. Phospholipase D (PLD) ()catalyses the
hydrolysis of phospholipids, resulting in formation of phosphatidic
acid (PtdOH) and liberation of the polar head group of the
phospholipid. PtdOH is further metabolized by PtdOH phosphohydrolase to
form diacylglycerol, an activator of protein kinase C. Data in human
neutrophils and rat mast cells have suggested that this could be the
major mechanism by which diacylglycerol accumulates during cell
activation(1, 2, 3, 4) . At least
two distinct isoenzymes of PLD are expressed in mammalian cells. One is
membrane-associated that prefers phosphatidylcholine as substrate,
whereas the other is a cytosolic enzyme that appears to hydrolyze
preferentially phosphatidylethanolamine or phosphatidylinositol
(PtdIns)(5, 6) . However, to date none of the PLDs
involved in signaling have been purified or cloned.
A large number of agonists activate PLD in mammalian cells(7, 8) . Although the mechanisms of enzyme activation, regulation, and the different functions of PLD remain largely unknown, several lines of evidence suggest that PLD is involved in various effector responses such as enzyme secretion(9, 10) , stimulation of the respiratory burst (11) , phagocytosis(12) , and cell proliferation(13, 14, 15, 16, 17, 18, 19) .
Protein kinase C activation by phorbol esters has been observed to
cause PLD activation, suggesting that PLD activation could be
downstream of the protein kinase C pathway. However, other mechanisms
of regulation of PLD activity that are protein kinase C-independent
have also been demonstrated. A guanine nucleotide binding protein
activated by guanosine 5`-O-3-(thio)triphosphate (GTPS),
can stimulate PLD activity(20, 21, 22) .
These studies suggest that a G protein directly interacts with PLD as a
upstream regulator. One G protein that regulates PLD activity has
recently been identified as ADP-ribosylation factor (ARF), a small
GTP-binding protein belonging to the Ras
superfamily(23, 24) . The effect of MgATP on PLD
activation is of particular interest. Although MgATP is not absolutely
required for PLD activation, it greatly potentiates the activation of
PLD by GTP
S (22, 25, 26, 27, 28, 29, 30) .
Because nonphosphorylating analogs of ATP do not possess the same
property, it appears that MgATP acts as a phosphoryl group donor in a
kinase-mediated phosphorylation reaction. The observation that
genistein, a tyrosine kinase inhibitor, significantly attenuated the
ability of MgATP to stimulate PLD activity in GTP
S-treated cells
led to the hypothesis that G proteins and tyrosine phosphorylation
could coordinately regulate PLD activity(29) . However indirect
evidence suggests that genistein is an inhibitor of
phosphatidylinositol 4phosphate 5-kinase (PtdIns 4P 5-kinase), the
enzyme responsible for the last step in the production of
phosphatidylinositol-4,5-bisphosphate
(PtdIns-4,5-P
)(31) . In addition, the
aminoglycoside antibiotic neomycin, which exhibits high affinity
binding to inositol phospholipids, inhibits PLD activity in vitro as well as in permeabilized cells(32) . These observations
suggested that MgATP activates PLD by acting as substrate for lipid
kinases.
Using partially purified brain membrane PLD assayed in
vitro, we have previously shown that PtdIns-4,5-P caused up to 10-fold increase in PLD activity in vitro in a highly specific manner(33) . Neomycin inhibited
membrane-bound PLD but had no effect on the activity of the partially
purified enzyme. The inhibitory effect of neomycin could be restored by
the addition of exogenous PtdIns-4,5-P
, suggesting that
neomycin inhibits membrane-bound PLD activity by binding to endogenous
PtdIns-4,5-P
.
In this study, we show that inhibition of
PtdIns-4,5-P synthesis potently inhibits PLD activation in
permeabilized U937 cells. Together with our previous results, these
findings indicate that PtdIns-4,5-P
is an essential
physiological cofactor of PLD.
Figure 1:
HPLC analysis of PtdIns-4-P and
PtdIns-4,5-P formation after GTP
S and MgATP
stimulation. Quiescent U937 cells were labeled overnight with myo-[
H]inositol and stimulated with 2
mM MgATP and/or 100 µM GTP
S during the
30-min permeabilization. The samples were analyzed by HPLC to separate
PtdIns-4-P from PtdIns-4,5-P
, which are presented
respectively in panelsA and B.
,
control;
, GTP
S;
, MgATP;
, GTP
S, and
MgATP. A representative result from separate experiments is
presented.
Figure 2: Anti-PtdIns 4-kinase antibody inhibits PtdIns 4-kinase activity. Lysates from quiescent U937 cells were incubated for 30 min at 4 °C in the presence of increasing concentration of antibody 4C5G, raised against the type 2 PtdIns 4-kinase. PtdIns 4-kinase activity was then assayed as described under ``Experimental Procedures.'' Unrelated control antibody showed no effect on this activity (not shown).
Figure 3:
Inhibition of PtdIns-4-P and
PtdIns-4,5-P synthesis by anti-PtdIns 4-kinase antibody in
permeabilized cells. Quiescent U937 cells were permeabilized for 30 min
in the presence of 100 µM GTP
S and 2 mM Mg[
-
P]ATP with or without 45 µg of
the antibody 4C5G. The permeabilization was carried out in 1 ml of
volume. Values are expressed as percent inhibition of PtdIns-4-P and
PtdIns-4,5-P
synthesis obtained in absence of inhibitor and
are averages of three experiments carried out in
triplicate.
Figure 4:
Inhibition of GTPS:MgATP-dependent
PLD activity by anti-PtdIns 4-kinase antibody. Cells were incubated in
permeabilization buffer with 100 µM GTP
S and 2 mM MgATP in the presence of the indicated concentration of the
monoclonal antibody 4C5G. An antibody recognizing the p85 subunit of
phosphoinositol 3-kinase was used as negative control. The reaction was
carried out for 30 min, and then PLD activity was measured as described
under ``Experimental
Procedures.''
Figure 5:
Inhibition of TPA-dependent PLD activity
by antiPtdIns 4-kinase antibody. The experiment was conducted as
described under Fig. 4with the difference that TPA was added as
stimulus (in the absence of exogenous MgATP and GTPS). The
antibody 4C5G was included in the assay at a concentration of 45
µg/ml.
Figure 6:
Inhibition of GTPS:MgATP-dependent
PLD activity by neomycin. Cells were incubated in permeabilization
buffer with 100 µM GTP
S and 2 mM MgATP with
increasing concentration of neomycin. PLD activity was described as
described above.
Figure 7:
PtdIns-4,5-P effects on PLD
activity. The PLD activity in total cell lysate was measured in the
presence of increasing concentration of PtdIns-4,5-P
as
described under ``Experimental
Procedures.''
PtdIns-4,5-P is an important component of several
intracellular signaling pathways. It is the precursor of the second
messengers inositol trisphosphate and diacylglycerol; in addition, it
serves as a substrate for phosphoinositide 3-kinase yielding
phosphatidylinositol 3,4,5-trisphosphate, a messenger that has recently
been shown to activate Ca
-independent protein kinase
C isozymes(42, 43) . PtdIns-4,5-P
itself
may directly bind to and modulate the activity of multiple proteins,
and this interaction may coordinate and regulate a number of different
processes within the cell. Thus, in addition to its established
precursor role in signal transduction, PtdIns-4,5-P
may
mediate effects of different stimuli on actin polymerization and in
general on cytoskeleton organization. In this context,
PtdIns-4,5-P
has been shown to release gelsolin and other
capping proteins from actin filaments, release actin monomers from
profilin, stimulate
-actinin-dependent actin bundling, and
activate the GTPase activity of
dynamin(44, 45, 46, 47, 48, 49, 50, 51) .
We have recently shown the importance of PtdIns-4,5-P as
a cofactor for the membrane-bound PLD and the partially purified
enzyme. The present paper demonstrates the physiological relevance of
PtdIns-4,5-P
as cofactor for PLD activation in
permeabilized U937 cells stimulated with GTP
S and MgATP. In these
cells(29) , as well as in several other cell types (for review,
see (7) ), the activation of PLD by GTP
S is greatly
potentiated by MgATP. We hypothesized that MgATP potentiated PLD
because it is required as a substrate for phosphoinositide kinases that
produce PtdIns-4,5-P
as a PLD cofactor. The data presented
here support this hypothesis. First, the presence of MgATP was found to
be essential for maintaining elevated phosphoinositide levels in the
cells both in the absence and presence of GTP
S (Fig. 1).
Second, the synthesis of PtdIns-4-P and PtdIns-4,5-P
(in
the presence of MgATP and GTP
S) was inhibited by an inhibitory
antibody specific for PtdIns 4-kinase ( Fig. 2and Fig. 3), indicating that MgATP supported on-going
PtdIns-4,5-P
synthesis in the permeabilized cells. Third,
the anti-PtdIns 4-kinase antibody inhibited PLD activation ( Fig. 4and Fig. 5), and, in addition, adenosine, an agent
that inhibits type 2 PtdIns 4-kinase by competing with
ATP(52) , inhibited by 50% MgATP:GTP
S-dependent PLD
activity at the concentration of 5 mM (where the concentration
of MgATP used was 0.5 mM (data not shown)), indicating that
on-going PtdIns-4,5-P
synthesis/high cellular
PtdIns-4,5-P
levels are required for PLD activity under
these conditions. These evidences, and the ability of neomycin, a high
affinity ligand of PtdIns-4,5-P
, to block the
MgATP:GTP
S stimulation of PLD, supports this conclusion (Fig. 6). Finally, it was demonstrated that PtdIns-4,5-P
can potently stimulate PLD activity in a Triton X-100-based
lysate of U937 cells in vitro (Fig. 7).
These data
clearly show that PtdIns-4,5-P is required for PLD
activation and are consistent with our hypothesis that it functions as
a cofactor of PLD. However, because under the present conditions
GTP
S would activate phospholipase C-
(53) , an
alternative explanation for the data is that PtdIns-4,5-P
is required as a precursor of the protein kinase C activator
diacylglycerol and that the activation of PLD by GTP
S is secondary
to activation of phospholipase C-
and protein kinase C. This
explanation is inconsistent with the fact that the anti-PtdIns 4-kinase
antibody inhibits by 60% PLD activation by the phorbol ester TPA (Fig. 5), since TPA activates protein kinase C directly,
bypassing phospholipase C-
and diacylglycerol, and obviates the
need for PtdIns-4,5-P
in its capacity as a precursor.
Furthermore, under conditions of protein kinase C activation, the
activity of phospholipase C-
is subject to negative feedback
inhibition(54) . It is interesting to note that a fraction
(
40%) of protein kinase C-induced stimulation of PLD activity is
not sensitive to inhibition by anti-PtdIns 4kinase antibody, whereas
GTP
S-induced activation was completely inhibited. This may
indicate that activation of PLD by protein kinase C abrogates in part
the cofactor requirement of PLD for PtdIns-4,5-P
, whereas
activation of PLD via G protein does not. Indeed, it has been shown
recently that activation of PLD by the small G protein ARF is
absolutely dependent on the presence of
PtdIns-4,5-P
(24) .
The identification of ARF, a
small G protein that belongs to the ras superfamily, as an
activator of PLD in myeloid cells (23, 24) has
strongly implicated PLD and its product PtdOH in vesicular traffic. The
function(s) of PLD and PtdOH in vesicular traffic are, however,
currently unknown. The identification of PtdIns-4,5-P as a
cofactor for PLD ( (33) and the present paper) may provide an
answer to this question. Evidence that polyphosphoinositide synthesis
is important for membrane transport events has been emerging in recent
years (for review, see (55) ). Thus, the process of PLD ( (33) ARF and the biosynthesis of PtdIns-4,5-P
may
participate in a coordinate mechanism for membrane vesiculation and/or
fusion. We have recently proposed a model whereby the formation of
PtdOH and PtdIns-4,5-P
, by PLD and PtdIns-4-P 5-kinase,
respectively, participate in a positive feedback loop that may play an
important role in vesicle fusion with acceptor membranes(33) .
Briefly, it was suggested that the interaction of coated vesicles
bearing ARF
GTP (56) with acceptor membranes will activate
PLD associated with these membranes, producing
PtdOH(23, 24) . The activity of PtdIns-4-P 5-kinase
(which is hypothesized to be located at acceptor membranes) will be
stimulated by PtdOH(57, 58) , resulting in massive
synthesis of PtdIns-4,5-P
from vesicular PtdIns-4-P. This,
in turn, will cause further stimulation of PLD activity ( (33) and the present study) leading to more PtdOH production
and further stimulation of PtdIns-4-P 5-kinase, etc. This will rapidly
cause a very profound change in the lipid composition of vesicular
membranes, leading to the formation of microdomains, which are greatly
enriched in PtdOH and PtdIns-4,5-P
. The activity of ARF
GTPase-activating protein is stimulated synergistically by
PtdIns-4,5-P
and PtdOH(59) . Thus, the
PtdIns-4,5-P
/PtdOH-rich vesicle membranes will cause ARF
GTPase-activating protein activation, stimulation of the GTPase
activity of ARF, and the conversion of active ARF
GTP to inactive
ARF
GDP. Consequently, PLD will be de-activated, thus halting the
positive feedback loop, initiating the disassembly of the coated
vesicle(60) , and allowing its subsequent fusion with acceptor
membranes (see (33) for more details).
One prediction of
this model is that in membrane traffic the activation of PLD and the
stimulation of PtdIns-4,5-P biosynthesis are tightly
coupled. Indeed, the present results indicate that ongoing
PtdIns-4,5-P
synthesis is required for PLD activation by G
protein in U937 cells. A corollary of this prediction is that
PtdIns-4-P 5-kinase is a major physiological target of PtdOH.
Intriguingly, in the presence of MgATP, the synthesis of
PtdIns-4,5-P
is greatly potentiated by GTP
S (Fig. 1). This result may suggest that the enzyme responsible
for PtdIns-4-P and PtdIns-4,5-P
are themselves regulated by
G protein(s). This idea was previously suggested on the basis of
GTP
S effects on isotope flux from PtdIns to IP
in
permeabilized fibroblasts(61) . An alternative explanation is
that, as predicted by our model, the biosynthesis of PtdIns-4,5-P
is stimulated by PLD-produced PtdOH. Further studies are required
in order to elucidate the mechanism of activation of PtdIns-4,5-P
synthesis by GTP
S.
The effects of the monoclonal antibody 4C5G demonstrate its efficacy as a tool in functional studies on phosphoinositide metabolism. The inhibition of PtdIns 4-kinase activity by the antibody 4C5G could be extremely useful in permeabilized or microinjected cells in experiments designed to clarify the influence of PtdIns 4-kinase and its products on different pathways. It is clear that multiple PtdIns 4-kinases and PtdIns-4-P 5kinases exist in mammalian cells(62) , but thus far only a single cDNA clone for a mammalian PtdIns 4-kinase has been isolated(63) . Once specific molecular tools are available for studies of phosphoinositide kinases, the proposed relationship between these enzymes and PLD in membrane traffic could be explored in greater depth.