(Received for publication, July 18, 1995)
From the
Phospholipase D (PLD) activation by guanine nucleotides requires
protein cofactors from both the membrane and the cytosol. The small
GTP-binding protein ADP-ribosylation factor (ARF) has been established
as one important component of PLD activation. By stimulating HL-60
cells with various agonists and then isolating the membrane fraction
and assaying PLD activity in the presence and absence of GTPS, we
observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a
potentiation of GTP
S-stimulated PLD activity in the membrane
fractions of these cells. Inactive phorbol esters induced no such
potentiation. Both fMLP and active phorbol esters induced a
2-3-fold increase in GTP
S-stimulated PLD in HL-60 membranes.
Membranes derived from stimulated HL-60 cells contained 60-70%
more ARF as compared with membranes derived from control cells.
Membrane contents of ARF were assessed by Western blotting with the
anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation.
Therefore, ARF translocation correlates with the potentiation of the
GTP
S-stimulated PLD activity. The effect on PLD activity and ARF
membrane content achieved through fMLP stimulation was greatly enhanced
by prior treatment of the cells with cytochalasin B. Membranes derived
from control and fMLP-stimulated cells were assayed for PLD activity in
the presence of exogenous ARF and a 50-kDa fraction known to contain
elements implicated in PLD activation. The ability of ARF and the
50-kDa fraction to enhance GTP
S-sensitive PLD activity was
significantly reduced when the membranes were derived from
fMLP-stimulated cells. The data indicate that, in addition to ARF,
elements of the 50-kDa PLD-inducing factors were likely already
translocated to the membranes upon stimulation. We propose that ARF,
upon stimulation with agonists such as fMLP or phorbol esters, is
translocated to the membrane and in concert with other protein
components of the 50-kDa fraction enhances PLD activity.
Neutrophils exercise their functions in primary host defense
against pathogens through different effector systems including
superoxide generation and granular secretion. It is now generally
accepted that phospholipases are implicated in the generation of second
messengers essential to these effector systems(1) .
Phospholipase C catalyzes the hydrolysis of phosphatidylinositol
4,5-biphosphate into inositol 1,4,5-triphosphate and diradylglycerol;
two important second messengers. PLD ()hydrolyzes
phosphatidylcholine into choline and phosphatidic acid. Phosphatidic
acid is possibly involved in superoxide anion production by inducing
the dissociation of the Rac-GDP/GTP dissociation inhibitor
complex(2) , thereby releasing Rac, which can then assemble the
NADPH oxidase complex (e.g. for review see (3) ).
Phosphatidic acid can also be converted to diradylglycerol by the
enzyme phosphatidic acid phosphatase. Diradylglycerol derived from the
PLD and phospholipase C pathways has been shown to cause different
patterns of protein kinase C isoform translocation and to have distinct
cellular effects(4) . Thus PLD may play an important role in
neutrophil cell physiology.
Many agonists are known to stimulate PLD activity in neutrophils including fMLP, interleukin-8, C5a, leukotriene B4, and platelet activating factor(5, 6, 7, 8) . The receptors for these agonists are composed of seven transmembrane segments and are coupled to heterotrimeric G-proteins (for a review see (9) ), which in turn can initiate a cascade of signaling events that lead to PLD activation. Accordingly, direct activation of G proteins by nonhydrolyzable analogues of GTP stimulates the activation of PLD in cell-free systems. PLD is known to be activated by at least two distinct pathways: a tyrosine kinase-mediated pathway and a protein kinase C-mediated pathway (10, 11, 12, 13) . Stimulation of PLD by agonists such as fMLP is pertussis toxin-sensitive, but activation of protein kinase C by phorbol esters induces a PLD response that is pertussis toxin-insensitive(14, 15) . Increasing tyrosine phosphorylation by use of inhibitors of tyrosine phosphatases also causes a pertussis toxin-insensitive activation of PLD. Therefore PLD must lie downstream of a heterotrimeric G protein and be coupled to protein kinase C as well as to the tyrosine phosphorylation pathways.
Protein kinase C isoenzymes have been
reported to stimulate a membrane-associated PLD activity(16) .
Synergistic activation of PLD by guanine nucleotides was observed in
rabbit platelet membranes when cells were pretreated with protein
kinase C activators(17) . The response was not mimicked by
exogenously added protein kinase C, suggesting that the synergism might
be the result of the translocation of cytosolic components to the
membrane, presumably GTP-binding proteins. It was recently discovered
that a small GTP-binding protein named ADP-ribosylation factor (ARF)
stimulated PLD in in vitro assays using a reconstituted system
including partially purified PLD and exogenously added ARF and
GTPS(18, 19) . ARF was first identified as a
cofactor necessary for the ADP-ribosylation of subunit
G
by cholera toxin and classified as a G-protein in
its own right, belonging to the Ras superfamily of small GTP-binding
proteins(20) . ARF is myristoylated on the amino-terminal Gly
residue(18) . Myristoylation appears to be essential for ARF
functional activity (for a review see (21) ). ARF is required
for coatomer assembly on the Golgi, promoting formation and fusion of
non-clathrin-coated vesicles(22, 23) . Upon
stimulation with various agonists, including phorbol 12-myristate
13-acetate (PMA), ARF is translocated to the Golgi membrane in Hela
cells(24) .
In the present study we demonstrate that the
treatment of intact HL-60 cells with a phorbol ester, PMA, or the
chemotactic peptide, fMLP, induces a potentiation of the
GTPS-stimulated PLD in membrane fractions. Using ARF-specific
antibodies, we found that ARF translocates to membranes in response to
physiological or nonphysiological agonists, paralleling that of PLD
activity. These results document a strong correlation between ARF
translocation and PLD activation. Such translocation may play major
roles in the mechanisms governing the activation of PLD.
In some experiments partially purified 50-kDa PLD-inducing fractions were prepared as described previously by Bourgoin et al.(25) . Recombinant ARF was produced and purified as described(27) .
Figure 1:
Potentiation of GTPS-dependent PLD
activity by pretreatment of HL-60 with PMA and fMLP time dependence.
Labeled intact HL-60 cells were pretreated for 100 nM PMA (A) or incubated for 5 min with 10 µM CB and
stimulated with 0.1 µM fMLP (B) for the indicated
times. Membranes were prepared, and PLD activity was assayed in the
presence (
) and absence (
) of 20 µM GTP
S
for 20 min at 37 °C as described under ``Experimental
Procedures.'' There were no stimulations at time 0. Values are
mean ± S.E. of at least three experiments. *, p <
0.05 for values compared with the control using Student's paired t test.
The biological
effects of the chemotactic peptide fMLP include the activation of
PLD(7, 15) . Therefore, the effects of fMLP on the
activity of the membrane-associated PLD were examined next. HL-60 cells
were preincubated for 5 min with CB and then stimulated with fMLP for
varying times, following which the membrane fractions were prepared.
The PLD activity was then assayed in the absence or presence of 20
µM GTPS for 20 min (Fig. 1B). The
basal membrane-associated PLD activity (i.e. in the absence of
GTP
S) remained essentially stable during the length of the
experiment and was affected only slightly, if at all, by a
preincubation of HL-60 cells with fMLP. On the other hand, the
magnitude of the response to GTP
S rapidly increased in membranes
from cells stimulated with the formylated peptide. The effect of fMLP
was observed after 15 s and reached a maximum within the first minute
of preincubation. The levels of PEt produced in response to GTP
S
were maintained for up to a 5-min preincubation with fMLP.
In view of the role that has been
postulated for ARF in the regulation of PLD activity, we then sought to
examine the amounts of ARF in membrane fractions derived from untreated
HL-60 cells and from phorbol ester-treated cells. Membranes from
untreated HL-60 cells as well as from cells treated for 2.5 min with
PMA, PdBu, or 4PdBu were prepared and blotted with anti-ARF
antibodies. The blots (Fig. 2B) were analyzed by
densitometry, and the results of these experiments are summarized in Fig. 2A. These data demonstrate detectable amounts of
ARF in membranes from resting cells. In addition, the levels of
membrane-bound ARF were significantly increased in response to the
protein kinase C activators PMA and PdBu but not by 4
PdBu. When
expressed as a percentage of the unstimulated level, the amount of
membrane-associated ARF averaged 163 ± 8%, 153 ± 13%, and
97 ± 21% (n = 4) in response to PMA, PdBu, and
4
PdBu, respectively.
Figure 2:
Evaluation of membrane-associated ARF upon
stimulation of HL-60 cells with phorbol esters and fMLP. Intact HL-60
cells were treated with 100 nM PMA, 250 nM PdBu, 250
nM 4PdBu, 0.1 µM fMLP, or Me
SO
for 2.5 min. The membrane content of ARF was determined by resolution
of 100 µg of protein samples on 12% SDS-polyacrylamide gel
electrophoresis, electrotransfer to polyvinylidene difluoride membrane
and immunostaining with the anti-ARF monoclonal antibody 1D9 as
described under ``Experimental Procedures.'' A,
densitometric evaluation of membrane-associated ARF in phorbol
ester-treated cells. B, a blot representative of four
independent experiments is shown. C, densitometric evaluation
of membrane-associated ARF in fMLP-treated cells. Intact cells were
incubated for 5 min at 37 °C with 10 µM CB and
stimulated for 1 min with fMLP. D, a blot representative of
four similar experiments is presented.
Consistent with the prior observations with phorbol esters, preincubation of HL-60 cells with fMLP 100 nM for 1 min also enhanced the amount of membrane-associated ARF, as can be observed after a SDS-polyacrylamide gel electrophoresis resolution of membrane proteins and Western blotting with the anti-ARF antibody 1D9 (Fig. 2D). Densitometric evaluation of blots statistically confirmed that fMLP induced a translocation or association of cytosolic ARF to the membranes. In six independent experiments, the amount of membrane-bound ARF averaged 165 ± 10% as compared with control membrane fractions, when fMLP was the stimulus (Fig. 2C)
Figure 3:
Effect of CB on ARF translocation and PLD
activity induced by fMLP and PMA. Labeled or unlabeled intact HL-60
cells were either pretreated for 5 min with 10 µM CB or
the vehicle (MeSO) and stimulated for 2.5 min with 100
nM fMLP and 100 nM PMA alone or in combination.
Membrane fractions were prepared as described under ``Experimental
Procedures.'' A, ARF content was determined by resolution
of 30-µg protein samples on 12% SDS-polyacrylamide gel
electrophoresis, electrotransfer to polyvinylidene difluoride membrane
and immunostaining using an anti-ARF polyclonal antibody. A blot
representative of two experiments is shown. B, PLD activity
was assayed with or without 20 µM GTP
S for 20 min at
37 °C. Values are mean ± S.E. of at least three experiments. p values were obtained using Student's paired t test.
Figure 4:
Effects of exogenous recombinant ARF or
the 50-kDa factor on PLD activity in membranes derived from control or
fMLP-stimulated cells. Labeled intact HL-60 cells were pretreated for 5
min with 10 µM CB and stimulated with either 100 nM fMLP for 2 min or with the vehicle (MeSO). Membrane
fractions were prepared, and PLD activity was assayed in the presence
and absence of ARF or the 50-kDa fraction. A, where indicated,
10 µg of proteins from the 50-kDa fraction were incubated with or
without 20 µM GTP
S. B, where indicated, 1
µM myristoylated recombinant ARF was incubated in the
presence and absence of GTP
S for 20 min at 37 °C. Values are
mean ± S.E. of at least three
experiments.
The addition of ARF to control membranes in the
presence of GTPS increases PLD activity to levels similar to that
of membranes derived from fMLP-stimulated cells. By contrast, the
addition of ARF to membranes derived from fMLP-treated cells has little
or no effect on PLD activity induced by GTP
S (Fig. 4B). In the absence of GTP
S, ARF was without
effect on basal PLD activity.
PLD activation requires both cytosolic and membrane
factors(29, 30) . Activation of PLD by GTP analogues
implies a role for GTP-binding proteins. In granulocytes and HL-60
cells, GTP analogues do not stimulate PLD activity in cytosol-depleted
cells. A small GTP-binding protein, ARF, has been identified as an
important component in PLD activation. The addition of exogenous
recombinant ARF is able to reconstitute GTPS-stimulated PLD
activity in cytosol-depleted human granulocytes(19) . Though
GTP
S-dependent PLD has not been purified to homogeneity, an
ARF-dependent PLD activated by ARFs has been
characterized(18, 31) . Evidence also suggest that
PMA-mediated responses may be dependent on the presence of
ARF1(32) . The observation that PMA-pretreatment greatly
enhances the ARF-dependent PLD activity suggests the recruitment of
additional cytosolic components to the membranes.
The present study
was undertaken to determine whether translocation of cytosolic ARF to
the membranes is required for maximum PLD activation and if distinct
PLD activation pathways, protein kinase C-mediated and
receptor-mediated, had a similar downstream effect, which is the
association of cytosolic ARF to the membrane fraction. We demonstrated
that a pretreatment of intact HL-60 cells with fMLP or PMA enhances the
subsequent response to GTPS by 2-3-fold in isolated membrane
fractions, suggesting the stable recruitment of cytosolic components at
the membranes. The GTP dependence of the effects suggests translocation
of GTP-binding proteins. This is in agreement with the observation that
the addition of PMA to isolated membrane fractions had no effect on
GTP
S-stimulated PLD activity (results not shown). Following
pretreatment of intact HL-60 cells with two different types of agonists
we demonstrated, by Western blotting and densitometric analysis of
blots, a 60-70% increase in the levels of membrane-bound ARF.
When either PMA or fMLP are used as agonists, similar downstream
effects can be observed: the binding of ARF to the membranes and
potentiation of the responses to GTP
S. Treatment of rat basophils
with phorbol esters was similarly reported to promote ARF binding to
the Golgi membrane(24) . Maximal stimulation of membrane
GTP
S-sensitive PLD as well as ARF translocation induced by fMLP
required a prior treatment with CB. CB was shown to have no effect on
either PLD activity or ARF translocation when used on control cells or
PMA-stimulated cells. Our results differ from those of Balsinde et
al.(33) in that in the cell-free system no synergy was
observed between PMA and fMLP. In fact, when both agonists were used
together PLD activity was no higher than when the cells were stimulated
by a single agonist.
As described previously by
Cockcroft(34) , we found that the incubation of membrane
fractions derived from PMA-pretreated cells with
NH-terminal peptides of ARF1-(2-17) and
ARF4-(2-17) inhibited GTP
S-stimulated PLD activity in
membrane fractions by approximately 50%, while unrelated peptides such
as Rab3A and G
peptides had no effect on GTP
S
responses. Though ARF peptides may have nonspecific
effects(34) , the data are consistent with the peptide
ARF1-(2-17) being a specific inhibitor in an ARF
assay(35) .
Accumulating evidence demonstrates that PLD
activation required multiple cytosolic-derived components, including
ARF-type small GTPases and other factors contained in a 50-kDa
cytosolic fraction(25, 36) . The cytosolic factors
implicated in PLD activation have been further investigated by Singer et al.(37) and are now thought to comprise at least
two different groups of factors implicated in PLD activation apart from
ARF. One group contains RhoA and is GTPS-sensitive, and the other
group activates PLD, albeit slightly, in the absence of GTP
S.
These investigators and others also demonstrated that PLD activity is
synergistically activated by ARF and
RhoA(36, 37, 38) . Our results show that
stimulation of cells by fMLP renders their membrane fractions less
sensitive to activation by exogenous ARF or the 50-kDa cytosolic
factors, suggesting that these factors have already been translocated
to the membrane. However, the magnitude of PLD activation in membrane
from control and to a lesser extent from stimulated cells depends
largely on exogenously added 50-kDa fraction. The 50-kDa fraction was
shown to contain Rho-related proteins and cytosolic factor(s) other
than RhoA and Rac2(25) . A faint translocation of RhoA was
observed in membranes derived from fMLP-stimulated cells in the
presence of CB exclusively and not with PMA alone or in combination
with fMLP (data not shown). The data suggest that component(s) of the
50-kDa PLD-inducing fraction are also translocated to the membrane;
however, their association may be less stable than that observed with
ARF. The amounts of membrane-associated 50-kDa factors is likely the
rate-limiting step for maximal PLD activation in membranes derived from
stimulated cells.
Maximal activation of the GTP-dependent PLD requires lipid cofactors such as phosphatidylinositol 4,5-biphosphate and possibly phosphatidylinositol 3,4,5-triphosphate(18, 39, 40) . The phosphatidylinositol 4,5-biphosphate dependence is consistent with results showing that acidic phospholipids have complex effects on ARF and ARF-regulatory proteins(41, 42) . Thus, phosphatidylinositol lipids resynthesis may act in concert with ARF in a general mechanism for PLD activation. Accordingly, the activation of PLD by neutrophil agonists has been reported to be inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase(43, 44) . RhoA may have a role in PLD activation through stimulation of phosphatidylinositol 3-kinase (45) and phosphatidylinositol 4-phosphate 5-kinase(46) .
In summary, ARF and possibly other factors
such as those contained in the 50-kDa fraction are translocated to the
membrane fraction of HL-60 cells when these cells are pretreated with
either an active phorbol ester or the chemotactic peptide fMLP. ARF
translocation correlates with increased GTPS-stimulated PLD
activity in isolated membranes. An essential role for cytosolic ARF in
PLD activation is supported by the present study. Cytosolic ARF needs
to be associated with the membrane to be active and to interact with
its effector, possibly membrane-bound PLD itself. It will be
interesting to see if ARF interacts directly with PLD or through the
assembly of a multifunctional protein complex upon stimulation of HL-60
cells with appropriate agonists.