(Received for publication, March 29, 1995; and in revised form, August 28, 1995)
From the
An activator of rat brain phospholipase D (PLD) that is distinct
from the already identified PLD activator, ADP-ribosylation factor
(ARF), was partially purified from bovine brain cytosol by a series of
chromatographic steps. The partially purified preparation contained a
22-kDa substrate for Clostridium botulinum C3 exoenzyme
ADP-ribosyltransferase, which strongly reacted with anti-rhoA
p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21
antibody. Treatment of the partially purified PLD-activating factor
with both C3 exoenzyme and NAD significantly inhibited the
PLD-stimulating activity. These results suggest that rhoA p21
is, at least in part, responsible for the PLD-stimulating activity in
the preparation. Recombinant isoprenylated rhoA p21 expressed
in and purified from Sf9 cells activated rat brain PLD in a
concentration- and GTPS (guanosine
5`-O-(3-thiotriphosphate))-dependent manner. In contrast,
recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed
to activate the PLD. This difference cannot be explained by a lower
affinity of non-isoprenylated rhoA p21 for GTP
S, as the
rates of [
S]GTP
S binding were very similar
for both recombinant preparations and the GTP
S-bound form of
non-isoprenylated rhoA p21 did not induce PLD activation.
Interestingly, recombinant isoprenylated rhoA p21 and ARF
synergistically activated rat brain PLD; a similar pattern was seen
with the partially purified PLD-activating factor. The synergistic
activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of
recombinant isoprenylated rhoA p21 in a NAD-dependent manner.
Inhibition correlated with the extent of ADP-ribosylation. These
findings suggest that rhoA p21 regulates rat brain PLD in
concert with ARF, and that isoprenylation of rhoA p21 is
essential for PLD regulation in vitro.
Phospholipase D (PLD) ()catalyzes the hydrolysis
primarily of phosphatidylcholine (PC) to produce choline and
phosphatidic acid (PA)(1, 2, 3) . In the
presence of ethanol, however, PLD produces phosphatidylethanol (PEt) by
a transphosphatidylation reaction. The latter product provides an
unambiguous marker for assay of
PLD(1, 2, 3) . Using this assay, PLD
activation upon agonist stimulation has been demonstrated in many types
of mammalian cells and tissues (see (4) and (5) and
references therein).
It has been suggested that protein kinase C, calcium ion, pertussis toxin-sensitive and -insensitive GTP-binding proteins, and protein tyrosine kinase are involved in agonist-stimulated PLD activation (see (4) and (5) and references therein; see also (6) and (7) ). Of these putative PLD regulators, a member of the family of low molecular weight GTP-binding proteins (small G proteins), ADP-ribosylation factor (ARF), has been identified as a PLD activator by two independent research groups(8, 9) . Brown et al.(8) have reconstituted PLD partially purified from plasma membranes of HL-60 cells with purified ARF. Cockcroft et al.(9) have employed streptolysin O-permeabilized HL-60 cells as a PLD source for reconstitution with purified ARF. In addition to the PLD of HL-60 cells, activation by ARF has also been documented with partially purified rat brain PLD(10) . Thus, PLDs of HL-60 cells and of rat brain are directly or indirectly regulated by ARF.
Another member of the small G protein family, rhoA p21, has been identified by Malcolm et al.(11) as a PLD activator in studies on rat liver membranes. Rat liver PLD, however, was found not to be activated by ARF(11) . These and the preceding results suggest that there are different isoforms of PLDs and that they are regulated by distinct mechanisms. Massenburg et al.(10) have provided support for this concept, demonstrating that rat brain membranes contain two isoforms of PLD, one of which is ARF-sensitive and another oleate-sensitive but ARF-insensitive. These reports raised the question whether rat brain PLD, like rat liver PLD, might also be activated by rhoA p21.
In the process of confirming ARF activation of partially purified rat brain PLD, we have found that bovine brain cytosol contains a PLD-activating factor that is distinct from ARF and may be rhoA p21. In the present study, we show directly that rhoA p21 can also activate rat brain PLD. Inconsistent with the observations of Malcolm et al.(11) , however, ADP-ribosylation of rhoA p21 by Clostridium botulinum C3 exoenzyme was found to interfere with its activation of rat brain PLD. Furthermore, it was demonstrated that rat brain PLD is synergistically activated by ARF and rhoA p21.
ARF was partially purified from bovine brain cytosol. Briefly, bovine brain was homogenized, filtered, and centrifuged as described above with rat brain. Proteins from the cytosol fraction were precipitated with ammonium sulfate at 25-70% saturation and dissolved in TEDP. After dialysis against TEDP, the proteins were subjected to DEAE-Sephacel column chromatography. ARF fractions eluted at 60 mM NaCl from the column were concentrated by Mini module (Asahi Kasei, Tokyo, Japan), and then applied onto a Sephacryl S-200 column that had been equilibrated with TEDP containing 50 mM NaCl and 1 µM GDP. The eluted ARF fractions were combined and concentrated by Amicon Centriprep.
Figure 1:
Mono Q column chromatography of the
PLD-activating factor of bovine brain cytosol. The PLD-activating
factor that is distinct from ARF was partially purified by
DEAE-Toyopearl 650S, phenyl-Sepharose CL-4B, and TSK-GEL 3000 SW XL
column chromatography (as described under ``Experimental
Procedures''). Fractions containing the PLD-stimulating activity
eluted from TSK-GEL 3000 SW XL column were subjected to Mono Q column
chromatography. Each fraction was reconstituted with the partially
purified rat brain PLD in the presence of 40 µM GTPS.
After incubation at 37 °C for 1 h, the PLD activity (
) was
determined by measuring [
H]choline formation (as
described under ``Experimental Procedures''). C3 exoenzyme
substrate in the fractions was analyzed by
[
P]ADP-ribosylation with C3 exoenzyme and
[
P]NAD (inset), as described under
``Experimental Procedures.''
Figure 3:
Effects of C3 exoenzyme and/or NAD on the
PLD-stimulating activity of partially purified PLD-activating factor.
The partially purified PLD-activating factor was incubated for 20 min
at 30 °C without or with 1 µg/ml C3 exoenzyme and/or 20
µM NAD, reconstituted with partially purified rat brain
PLD, and the PLD activity was determined by measuring
[H]choline formation (as described under
``Experimental Procedures''). Bars represent
differences between duplicate determinations in a typical
study.
Figure 7:
C3
exoenzyme-catalyzed ADP-ribosylation of recombinant rhoA p21
inhibits its ability to activate rat brain PLD synergistically with
ARF. Recombinant isoprenylated rhoA p21 (25 nM) was
incubated for 5 min at 30 °C without () or with 1 µg/ml C3
exoenzyme (
) in the presence of 25 µg/ml ARF. ARF alone (25
µg/ml) was also incubated without (
) or with C3 exoenzyme
(
) under the same conditions. The reaction mixtures included the
indicated concentrations of NAD. The pretreated small G proteins were
reconstituted with partially purified rat brain PLD, incubated for 10
min at 37 °C in the presence of 40 µM GTP
S and 1%
ethanol, and [
H]PEt formation was analyzed as
described under ``Experimental Procedures.'' For
[
P]ADP-ribosylation by C3 exoenzyme, 25 nM recombinant isoprenylated rhoA p21 was incubated with a
series of concentrations of [
P]NAD (4,000
cpm/pmol) for 5 min at 37 °C, then the extent of
[
P]ADP-ribosylation was determined by SDS-PAGE,
autoradiography, and Bioimage BAS2000 analysis (
). Maximal
[
P]ADP-ribosylation was achieved with 100
µM [
P]NAD. Bars represent
differences between duplicate determinations in a typical
study.
Figure 5:
[S]GTP
S
binding to recombinant rhoA p21s and effects of the
GTP
S-bound form of recombinant rhoA p21s on rat brain PLD
activity. A, recombinant isoprenylated (
) or
non-isoprenylated rhoA p21 (
) (10 nM) was
incubated for the indicated time at 37 °C with 1 µM
[
S]GTP
S.
[
S]GTP
S bound to rhoA p21s was
determined as described under ``Experimental Procedures.'' B, a series of concentrations of recombinant isoprenylated
(
) or non-isoprenylated rhoA p21 (
) were incubated
for 5 min at 37 °C with 80 µM GTP
S to effect
preloading, and reconstituted with rat brain PLD.
[
H]PEt production during a 1-h incubation at 37
°C was determined. Bars represent differences between in
duplicate determinations in a typical
study.
An activator of rat brain PLD that is distinct from the already identified PLD activator ARF was partially purified from bovine brain cytosol by a series of chromatographic steps (see ``Experimental Procedures''). The PLD-stimulating activity and C3 exoenzyme substrate of 22 kDa apparent molecular mass were co-purified during each step (data not shown). The final chromatography on Mono Q resulted in elution of both the PLD-stimulating activity and the C3 exoenzyme substrate as two peaks (Fig. 1). The major, second peak of PLD-stimulating activity from the Mono Q column contained a 22-kDa protein as assessed by SDS-PAGE (Fig. 2A). This protein strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody (Fig. 2B). The PLD-stimulating activity of the partially purified factor was suppressed upon incubation with both NAD and C3 exoenzyme, which specifically ADP-ribosylates and inhibits rho proteins (Fig. 3); no suppression occurred with either agent alone. These results, taken together, imply that rhoA p21 is an activator of rat brain PLD, in agreement with the finding of Malcolm et al.(11) .
Figure 2: Protein staining and immunoblotting of the partially purified preparation of PLD-activating factor probed with anti-rhoA p21, anti-rac1 p21, and anti-cdc42Hs p21 antibodies. Proteins in fraction 34 of the Mono Q column eluate containing the PLD-stimulating activity were analyzed by SDS-PAGE on 15% gels and Coomassie Brilliant Blue (CBB) staining (A). Immunoblots of the partially purified preparation of PLD-activating factor were probed with anti-rhoA p21, anti-rac1 p21, and anti-cdc42Hs p 21 antibodies (B), as described under ``Experimental Procedures.''
To confirm this hypothesis, isoprenylated rhoA p21 was expressed in and purified from Sf9
cells(13) , and reconstituted with partially purified rat brain
PLD. As shown in Fig. 4A, recombinant isoprenylated rhoA p21 activated in a concentration-dependent manner the rat
brain PLD in the presence of GTPS, but not in its absence. In
contrast, non-isoprenylated rhoA p21, expressed in and
purified from E. coli, failed to activate PLD (Fig. 4B). Recombinant isoprenylated and
non-isoprenylated rhoA p21s both bound
[
S]GTP
S with similar kinetics (Fig. 5A). Furthermore, recombinant non-isoprenylated rhoA that had been preloaded with GTP
S was also unable to
effect PLD activation (Fig. 5B). These findings rule
out a difference in binding of GTP
S as the explanation for the
difference in PLD activation between the two recombinant forms of rhoA p21. The results further indicate that rhoA p21
directly or indirectly activates rat brain PLD, and that
post-translational modification of rhoA p21, like
ARF(8, 9) , is essential for it to function as a PLD
activator.
Figure 4:
Effects of isoprenylated and
non-isoprenylated rhoA p21s on rat brain PLD activity.
Partially purified rat brain PLD was reconstituted with isoprenylated (A) and non-isoprenylated (B) rhoA p21s,
which were expressed in and purified from Sf9 cells and E.
coli, respectively. The reconstituted proteins were incubated at
37 °C without () or with 40 µM GTP
S
(
l) in the presence of 1% ethanol. [
H]PEt
production over 1 h was analyzed as described under ``Experimental
Procedures.'' Bars represent differences between
duplicate determinations in a typical
study.
Whereas the PLD in rat liver membranes is activated by rhoA p21 but insensitive to ARF(11) , rat brain PLD was activated by ARF (Fig. 6) as well as by rhoA p21 ( Fig. 4and 6). When the partially purified PLD-activating factor or recombinant isoprenylated rhoA p21 was reconstituted together with ARF, synergistic activation of rat brain PLD was observed (Fig. 6). Consistent with the result illustrated in Fig. 3, incubation of recombinant isoprenylated rhoA p21 with C3 exoenzyme plus a series of concentrations of NAD attenuated the synergistic activation of PLD in a NAD concentration-dependent manner (Fig. 7). The degree of inhibition correlated well with the extent of ADP-ribosylation of recombinant isoprenylated rhoA p21 by the C3 exoenzyme (Fig. 7).
Figure 6:
Synergistic activation of rat brain PLD by
ARF and the partially purified PLD-activating factor or isoprenylated rhoA p21. Partially purified rat brain PLD was reconstituted
without () or with (
) 1 µl of the PLD-activating
fraction obtained from the DEAE-Toyopearl 650S column (A) or
10 nM recombinant isoprenylated rhoA p21 (B)
in the presence of the indicated concentrations of ARF. After
incubation for 1 h at 37 °C in the presence of 40 µM GTP
S and 1% ethanol, [
H]PEt formation
was assessed (as described under ``Experimental
Procedures''). Bars represent differences between in
duplicate determinations in a typical
study.
Malcolm et al.(11) have recently reported that rhoA p21 activates rat liver PLD. We have shown in the present study that rat brain PLD is also activated by rhoA p21 (Fig. 4A). The mechanism for activation of rat brain PLD by rhoA p21, however, seems to be different from that of rat liver PLD. In particular, ADP-ribosylation of rhoA p21 by C3 exoenzyme perturbs the ability of rhoA p21 to activate rat brain PLD ( Fig. 3and Fig. 7), whereas rat liver PLD is still activated by ADP-ribosylated rhoA p21(11) .
Another difference between rat brain PLD (our observations) and rat liver PLD (data from Malcolm et al.) is the sensitivity of the PLD(s) to ARF; we found rat brain PLD to be activated by ARF (Fig. 6), whereas rat liver PLD is insensitive to ARF(11) . This supports the concept that the isoforms of PLD in rat liver and brain are distinct. A possible alternative is that rat brain contains two isoforms of PLD, one of which is ARF-sensitive and another rhoA p21-sensitive, whereas rat liver contains only the rhoA p21-sensitive PLD. However, this is unlikely. If there were two isoforms of PLD in rat brain, the activation of rat brain PLDs by ARF plus rhoA p21 would be expected to be additive. On the other hand, if rat brain PLD (considered a distinct isoform from that in rat liver) were regulated by both ARF and rhoA p21 by different mechanisms, activation by these agents might be synergistic. The results of the present study provide strong support for the latter possibility, as recombinant isoprenylated rhoA p21 and ARF activated rat brain PLD in a synergistic manner (Fig. 6). Thus, it is likely that rat brain PLD differs from rat liver PLD. Very recently, Siddiqi et al.(18) and Singer et al.(19) have reported that PLDs in membranes of HL-60 cells and porcine brain, respectively, are activated synergistically by ARF and rhoA p21. Their data suggest that the PLDs in membranes of HL-60 cells and porcine brain are analogous to that in rat brain membranes.
The PLD-stimulating activity partially purified from bovine brain cytosol was inhibited only incompletely (by about 50%) on ADP-ribosylation of rhoA p21 by C3 exoenzyme and NAD (Fig. 3). Although rac1 p21 and cdc42Hs p21 were not detected in this preparation (Fig. 2B), it is still possible that another C3 exoenzyme-insensitive PLD activator(s) contaminated the preparation. It has not proven practicable to assess whether exhaustive ADP-ribosylation of the preparation results in complete inhibition, because prolonged incubation (more than 20 min at 30 °C) of the partially purified PLD-activating factor drastically diminishes its activity even in the absence of C3 exoenzyme and NAD. Nevertheless, the results obtained (Fig. 3) imply that rhoA p21 is responsible, at least in part, for the PLD-stimulating activity of the preparation. Perturbation by C3 exoenzyme-catalyzed ADP-ribosylation of the ability of rhoA p21 to modulate rat brain PLD activity was clearly demonstrated in the studies employing recombinant rhoA p21 (Fig. 7). Although the rate of ADP-ribosylation of partially purified bovine brain rhoA was very slow (reaching a plateau after 60-120 min of incubation), ADP-ribosylation of the recombinant isoprenylated rhoA reached a maximum within 5 min under the conditions employed in Fig. 7(data not shown). Taking advantage of this characteristic of the recombinant rhoA, we were able to demonstrate that the extent of ADP-ribosylation of rhoA correlated well with the degree of inhibition of the synergistic activation of PLD by rhoA in concert with ARF (Fig. 7).
Synergistic activation of PLD by rhoA p21
and ARF has also been found with permeabilized, cytosol-depleted rabbit
neutrophils, suggesting that rabbit neutrophil PLD is
regulated by the same (or similar) mechanism as rat brain PLD. Lambeth et al. and Bourgoin et al. have recently reported
that ARF functions in concert with a 50-kDa cytosolic factor in the
activation of PLDs in human neutrophils and HL-60 cells,
respectively(20, 21) . In their studies, the molecular
weight of the cytosolic factor was estimated by gel filtration
chromatography. In the present study, the PLD-stimulating activity and
C3 exoenzyme substrate in the bovine brain cytosol co-chromatographed
on gel filtration at a position corresponding to
45 kDa (data not
shown). It has been established that rhoA p21 associates with
a Rho GTP/GDP exchange inhibitor, Rho GDI, with a molecular mass of 27
kDa(22) . Thus the complex of rhoA p21 with Rho GDI
should elute from the chromatograph with an apparent molecular mass of
50 kDa. Bourgoin et al.(21) found that rhoA p21 eluted with an apparent molecular mass of
45
kDa, but concluded that it is not the 50-kDa PLD-activating factor of
HL-60 cells as they failed to detect rhoA p21 in several
PLD-activating fractions. Although the
50-kDa PLD-activating
factor(s) in HL-60 cells and human neutrophils remains to be
identified, PLD in these cells may be regulated differently from that
in rat brain. Thus, the mechanism of PLD activation may vary between
one isoform of PLD and another.
Another finding in this study was
that, like ARF, rhoA p21 required post-translational
modification to effect its regulation of PLD: non-isoprenylated rhoA p21 failed to activate rat brain PLD, even in its
GTPS-bound state ( Fig. 4and Fig. 5B). It
has been reported that GTP-bound isoprenylated rhoA p21, but
not non-isoprenylated rhoA p21, translocates to plasma
membranes(23) . Consistent with the latter report, rhoA p21 (isoprenylated) in the partially purified preparation
of PLD-activating factor bound to phospholipids vesicles containing the
PLD substrate, PC, only in the presence of GTP
S, whereas
non-isoprenylated rhoA p21 did not bind under any
circumstances (data not shown). This result leads us to speculate that
the GTP
S-bound isoprenylated rhoA p21 also translocates
to the phospholipid vesicles, and thereby attracts PLD to the vesicles,
making the enzyme more accessible to its substrate and increasing
hydrolysis. Alternatively, PLD might interact specifically with
PIP
in the vesicles and the GTP
S-bound isoprenylated rhoA p21 translocate to the vesicles to interact with PLD. The
latter notion is based on the finding that translocation of
isoprenylated rhoA p21 to the vesicles does not require the
presence of PIP
, whereas PIP
is essential for
PLD activity (8) .
We are currently investigating
whether PLD interacts specifically with PIP
.