(Received for publication, September 15, 1995)
From the
We have recently demonstrated the existence of an ATP-activated
phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M. A.,
Balsinde, J., Dennis, E. A., and Insel, P. A.(1995) J. Biol.
Chem. 270, 11738-11740). We have now found that nuclear PLD
is synergistically activated by guanosine
5`-O-(thiotriphosphate) (GTPS) and ATP in a time- and
concentration-dependent manner, but these compounds do not alter the
sensitivity of the enzyme to activation by Ca
. The
synergistic stimulation of PLD activity could be blocked by addition of
the protein kinase C inhibitors chelerythrine and calphostin C.
Stimulation by GTP
S was abolished by guanosine
5`-O-(2-thiodiphosphate). Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating
effect of GTP
S on ATP-dependent nuclear PLD activity. Moreover,
use of the Rho GDP dissociation inhibitor to extract Rho family G
proteins from cell nuclei also inhibits PLD activity. Western blot
analyses of isolated nuclei revealed the presence of the small G
protein RhoA, but not of RhoB or the ADP-ribosylation factor.
GTP
S-stimulated ATP-dependent PLD activity could be reconstituted
in Rho GDP dissociation inhibitor-washed nuclei by addition of
recombinant prenylated RhoA, but not by addition of non-prenylated
RhoA. Taken together, these results indicate that nuclear PLD activity
is modulated via a RhoA-dependent activation that occurs downstream of
protein kinase C. Nuclear PLD, which appears to be a previously
unrecognized effector regulated by protein kinase C and G proteins, may
be involved in the regulation of nuclear function or structure.
The detailed mechanisms by which agonists that bind to
cell-surface receptors regulate nuclear events are inadequately
understood. A number of studies have provided compelling evidence for a
regulatory role of protein kinase C (PKC) ()in
agonist-regulated effects on nuclear function(1, 2) .
One mechanism for PKC action involves translocation of PKC to the
nucleus triggered by increased accumulation of nuclear diacylglycerol
(DAG)(1, 3) . The elucidation of a phosphoinositide
cycle located at the nucleus has suggested a means by which
extracellular stimuli might elicit nuclear responses via generation of
DAG (reviewed in (4) ). In addition, nuclear DAG may arise from
phospholipids other than the phosphoinositides, e.g. phosphatidylcholine(5) .
Phospholipase D (PLD) catalyzes the hydrolysis of cellular phospholipids, particularly phosphatidylcholine, in response to a variety of hormones, neurotransmitters, and growth factors(6) . Phosphatidic acid (PA), the primary lipid product of PLD, appears to possess growth factor-like properties and can act as a second messenger in certain cell types(7, 8) . Since PA can be dephosphorylated via PA phosphohydrolase to produce DAG, this represents an alternative pathway to that initiated by phosphoinositide-specific phospholipase C for increasing cellular DAG levels(9) . While DAG production by phosphoinositide hydrolysis occurs in an early and transient manner, DAG production through the PLD pathway can be delayed and prolonged, thereby allowing a sustained activation of PKC(10, 11) .
PLD activity can be regulated by multiple types of signals(6) . Both heterotrimeric and low molecular weight G proteins have been implicated in PLD activation. Recent evidence suggests a key role for the involvement of the low molecular weight G proteins ADP-ribosylation factor (ARF) and RhoA in agonist-induced PLD activation in certain cell types(12, 13, 14, 15) . Evidence for the involvement of heterotrimeric G proteins is less well established and relies primarily on the inhibition of PLD activation produced by treatment of several types of cells with pertussis toxin(16, 17) .
We have recently described that
nuclei from MDCK-D1 cells possess a PKC-regulated PLD activity that
seems to account for the bulk of DAG generated in the
nucleus(18) . In the present work, we demonstrate that nuclear
PLD activity is enhanced by nonhydrolyzable GTP analogs, in particular
by GTPS, and that this response to GTP
S is mediated by the
low molecular weight G protein RhoA. Our data suggest a novel mechanism
for regulation of nuclear PLD that involves the sequential actions of
protein kinase C and RhoA.
RhoGDI-treated nuclei were prepared as described previously(15) . Isolated nuclei were incubated with the indicated concentration of RhoGDI for 15-20 min at room temperature. The nuclei were centrifuged at 12,000 rpm for 1 min at 4 °C, and supernatants were kept for further analysis. When RhoA proteins were included, as indicated, nuclei were incubated for 5 min before initiation of the assay.
Figure 1:
Effect of GTPS on ATP-dependent
PLD activity from MDCK-D1 cell nuclei. Purified nuclei from
[
H]palmitic acid-labeled MDCK-D1 cells were
incubated in buffer for 5 min at 37 °C prior to addition of 1.5%
ethanol. The incubations proceeded for another 5 min prior to addition
of GTP
S (10 µM), ATP (500 µM), or
GDP
S (100 µM) as indicated.
[
H]PEt formation was quantitated as described
under ``Experimental Procedures'' and is expressed as
percentage of radioactivity in PEt with respect to total nuclear
phospholipids at each point. Values shown represent the mean ±
S.E. (n = 3). The experiment shown is representative of
three.
Figure 2:
Characterization of the GTPS effect
on PEt production in nuclei from MDCK-D1 cells. Experiments were
carried out as explained in the legend to Fig. 1. A,
concentration response of the GTP
S effect in the presence (
)
or absence (
) of 500 µM ATP; B,
concentration response of the ATP effect in the presence (
) or
absence (
) of 10 µM GTP
S; C, time
course of nuclear PEt accumulation; D, effect of varying the
free Ca
concentrations on nuclear PEt production in
the absence (
) or presence of 10 µM GTP
S
(
), 500 µM ATP (
), or 10 µM GTP
S plus 500 µM ATP (
). The incubations
in the absence of added Ca
received 2 mM EGTA. [
H]PEt formation is expressed as
percentage of radioactivity in PEt with respect to total nuclear
phospholipids in each point. Data are given as means ± S.E. from
triplicate determinations in representative experiments. Each set of
experiments was repeated at least three different times with similar
results.
A further characterization of the potentiating
effect of GTPS on ATP-induced PLD activation was carried out, and
the results are shown in Fig. 2. When the ATP concentration was
held constant (500 µM), addition of GTP
S resulted in
a concentration-dependent increase in PEt levels up to 1
µM, with half-maximal effects at
30 nM GTP
S (Fig. 2A). The effect of varying the ATP
concentration at a constant GTP
S concentration (10
µM) is shown in Fig. 2B. Optimal PLD
activation under these conditions was achieved at ATP concentrations
above 100 µM. GTP
S appeared to increase the apparent
affinity of ATP for activation of PLD since at an ATP concentration of
50 µM, which was by itself nonstimulatory, the presence of
GTP
S induced substantial PEt production (Fig. 2B).
Accumulation of PEt in the presence of GTP
S and ATP was linear for
15 min (Fig. 2C).
Ca has been
reported to be required for G protein-dependent activation of
PLD(13, 19, 22) . As shown in Fig. 2D, increasing the free Ca
concentration in the PLD assay from levels found in resting cells (0.1
µM) to those attained in stimulated cells (1
µM) (23) increased PLD activity under all
experimental conditions, i.e. basal, ATP-activated, and (ATP
+ GTP
S)-stimulated PLD activities. This demonstrated that
nuclear PLD is a Ca
-regulated enzyme. Therefore,
increases in intracellular Ca
as a result of cell
activation may play a role in modulating nuclear PLD. However, enhanced
enzyme activity was observed in nuclei exposed to resting
Ca
concentrations found in unstimulated cells.
Addition of ATP alone or together with GTP
S did not appear to
substantially increase the sensitivity of nuclear PLD activity to
Ca
.
Unlike Ca, Mg
was absolutely required for nuclear PLD activity stimulated by
either ATP or GTP
S plus ATP. No effect of ATP or GTP
S plus
ATP was detected on nuclear PLD in the presence of 1 mM EDTA.
Increasing the Mg
concentration in the assay linearly
increased PLD activity up to 5 µM Mg
,
with no further increases in activity at higher Mg
concentrations (data not shown).
When ATP was replaced by UTP
or the nonhydrolyzable analog AMP-PCP, AMP-PNP, or 2-methylthio-ATP, no
effect of GTPS on nuclear PLD was observed (Table 1).
Moreover, the potentiating effect of GTP
S on ATP-induced PLD
activity was completely abolished when assays were conducted in the
presence of the PKC inhibitors chelerythrine (EC
5
µM) and calphostin C (EC
0.4
µM) (Fig. 3). These data are consistent with the
conclusion that for GTP
S to increase nuclear PLD activity, an
ATP-driven phosphorylation reaction, apparently mediated by PKC, is
required.
Figure 3:
Effect of PKC inhibitors on PEt production
by MDCK-D1 cell nuclei. Nuclei isolated from
[H]palmitic acid-labeled MDCK-D1 cells were
assayed with different chelerythrine (A) or calphostin C (B) concentrations in the absence (
) or presence of 10
µM GTP
S (
), 500 µM ATP (
),
or 10 µM GTP
S plus 500 µM ATP (
).
Data points represent the mean ± S.E. (n = 3).
The experiment shown is representative of
three.
Figure 4:
Effect of C. botulinum C3
exoenzyme on PEt production by MDCK-D1 cell nuclei. A,
proteins (150 µg) from homogenates (H) or nuclei (N) were electrophoresed and immunodetected as described under
``Experimental Procedures.'' Antibodies against RhoA, RhoB,
or ARF were assayed, and the blots are shown. B, nuclei
isolated from [H]palmitic acid-labeled MDCK-D1
cells were treated in vitro with (striped bars) or
without (open bars) 10 µg/ml C3 exoenzyme of C.
botulinum. After this treatment, nuclei were incubated without
(control (Ctrl)) or with GTP
S (10 µM), ATP
(500 µM), or both as indicated. Data are given as means
± S.E. from triplicate determinations in representative
experiments. Each set of experiments was repeated at least four times.
*, p < 0.05 using Student's paired t test.
Rho
proteins can be ADP-ribosylated and inhibited by the C3 exoenzyme of C. botulinum(24) . To address the possible role of Rho
proteins in the regulation of nuclear PLD activity, we treated MDCK-D1
cell nuclei with C. botulinum C3 exoenzyme (10 µg/ml; 30
min). Such a treatment resulted in 75% inhibition of PEt
production by GTP
S plus ATP, suggesting a role for Rho in
regulating nuclear PLD activation (Fig. 4B). To obtain
further evidence for a role of Rho in the regulation of nuclear PLD
activity, we treated MDCK-D1 cell nuclei with RhoGDI, a protein that
interacts with post-translationally modified Rho proteins and extracts
them from membranes(25, 26) . When nuclei were treated
with increasing concentrations of RhoGDI and subsequently washed,
nuclear PLD became progressively less responsive to GTP
S (Fig. 5A). Western blot analysis confirmed that RhoA
was extracted from nuclei and appeared in the wash after RhoGDI
treatment (Fig. 5B). Thus, the inhibition of
GTP
S-stimulated nuclear PLD activity by RhoGDI was associated with
a decrease in RhoA in the nuclear preparations (Fig. 5, A and B).
Figure 5:
Inhibition of GTPS-stimulated PLD
activity in RhoGDI-treated nuclei. Nuclei isolated from
[
H]palmitic acid-labeled MDCK-D1 cells were
treated in vitro with the indicated concentrations of RhoGDI
and subsequently washed. A, PLD activity was measured in the
absence (
) or presence of 10 µM GTP
S (
),
500 µM ATP (
), or 10 µM GTP
S plus
500 µM ATP (
). Data are given as means ±
S.E. from triplicate determinations in representative experiments. Each
set of experiments was repeated at least three times. B,
proteins (50 µg) from nuclei (N) treated with or without 1
µM RhoGDI (as indicated) or supernatants (S) from
the subsequent washes were electrophoresed and immunodetected as
described under ``Experimental Procedures.'' Antibodies
against RhoA were assayed, and the blots are
shown.
In the next series of experiments, we used
recombinant RhoA to investigate whether addition of this protein to the
assay mixture could restore GTPS-stimulated PLD in RhoGDI-treated
nuclei. When prenylated RhoA was added to RhoGDI-treated nuclei, PEt
production in response to GTP
S was almost fully restored (Fig. 6). As a control, we also used non-prenylated RhoA, which
was not able to reconstitute the response (Fig. 6). This finding
is consistent with the notion that small G proteins from the ras family that are not modified post-translationally do not interact
efficiently with GDP/GTP exchange factors(27) .
Figure 6:
Reconstitution of GTPS-activated PLD
by RhoA in RhoGDI-treated nuclei. Nuclei isolated from
[
H]palmitic acid-labeled MDCK-D1 cells were
either untreated (control (Ctrl)) or treated with RhoGDI as
described under ``Experimental Procedures''. Five min before
initiating the reactions, 1.4 µM RhoA either prenylated (RhoA(p)) or non-prenylated (RhoA(np)) was added to
the nuclei, after which the reactions proceeded for 30 min in the
presence of 500 µM ATP and in the presence (striped
bars) or absence (open bars) of GTP
S. Addition of
RhoA proteins did not alter PEt production. Data are given as a
percentage of the response observed in ATP-treated nuclei in the
absence of GTP
S. Results are given as means ± S.E. of three
independent determinations. The data shown are representative of those
obtained in three independent experiments.
G proteins are widely recognized to play an essential role in signal transduction from many types of cell-surface receptors to effector proteins in the plasma membrane(23) . Evidence is emerging to suggest that G proteins may also play a similar role in transducing signals in the nucleus, perhaps at the nuclear membrane. In fact, both heterotrimeric and low molecular weight G proteins have been demonstrated to be localized in the nucleus (Fig. 5B)(28) . We have recently demonstrated the existence of a PKC-regulated PLD activity in nuclei from MDCK-D1 cells(18) . In the present work, we have demonstrated that activation of a G protein provides another means of regulating nuclear PLD activity.
The nuclear PLD activity found in MDCK cells differs
appreciably from other PLDs on the basis of its activation
characteristics. Several authors have described PLD activities in
cell-free systems that are not stimulated by ATP unless phorbol
12-myristate 13-acetate or GTPS is also present in the incubation
buffer(19, 29) . This is different from what we
observed with nuclear PLD activity from MDCK-D1 cells, in which ATP can
activate enzyme activity by acting as a phosphoryl donor apparently by
a PKC-driven phosphorylation reaction(18) . The data in the
present study suggest that activation by PKC is required for GTP
S
to increase nuclear PLD activity (Fig. 3). This implies that in
nuclei from MDCK-D1 cells, PLD regulation by G proteins must follow
protein phosphorylation by PKC. In resting cells, PKC is present in the
cytosol, and after stimulation of cells, PKC translocates to the
particulate fraction. However, it has been observed that individual PKC
isoenzymes appear to be restricted to particular intracellular loci
before stimulation. Thus, colocalization of some PKC isoenzymes with
their substrates might serve to ensure preferential and rapid
phosphorylation of these substrates after PKC activation. It has been
suggested that isoenzymes of PKC bind to specific anchoring proteins,
collectively termed receptors for activated protein kinase C, located
at various subcellular sites(30) . It is possible that these
proteins are involved in targeting PKC to the nucleus. The precise
substrate(s) of phosphorylation by PKC in the cascade of events leading
to activation of nuclear PLD is unclear. Possible substrate(s) include
G proteins that directly interact with PLD (perhaps increasing
interaction of the G proteins with the lipase and/or increasing the
rate of GDP/GTP exchange(31) ), an intermediate protein factor
such as a GTPase-activating protein (32) or a GDP dissociation
factor(33) , and/or PLD itself (perhaps allowing interaction
with G proteins).
Based on the inhibitory effects that C.
botulinum C3 exoenzyme and RhoGDI exert on activation of
ATP-dependent PLD by GTPS, our results support the involvement of
a member of the Rho family of small G proteins in the regulation of
nuclear PLD. More direct evidence in favor of this notion was obtained
with the experiments in which addition of recombinant RhoA to
RhoGDI-treated nuclei restored GTP
S-activated PLD. A Rho protein
has also been implicated in PLD regulation in the studies by Bowman et al.(14) in neutrophils and by Malcolm et al.(15) in rat liver. However, in neither of these studies
could the authors show PLD inhibition by C. botulinum C3
exoenzyme. The Rho family of low molecular weight G proteins is
composed of three variants of Rho proteins (A, B, and C) and two forms
of Rac proteins (Rac1 and Rac2) (34) . C. botulinum C3
exoenzyme specifically inhibits the Rho proteins, but not the Rac
proteins. This fact led Bowman et al.(14) to exclude
Rho as a candidate activator of neutrophil PLD, but the possibility
that in those studies, C. botulinum C3 exoenzyme did not
completely inhibit Rho or that the modified form still remained active
was not ruled out. Moreover, even though C. botulinum C3
exoenzyme failed to inhibit PLD activity in liver plasma membranes,
Malcolm et al.(15) also achieved reconstitution of
GTP
S-activated PLD activity by RhoA, thereby suggesting that RhoA
regulates PLD activity in liver.
Other studies have implicated ARF as a low molecular weight G protein that regulates PLD activity(12, 14) . Since ARF appears to be absent from MDCK-D1 cell nuclear preparations (but is present in homogenates), we believe it is unlikely that this G protein is a regulator of nuclear PLD activity in these cells. However, it is possible that other PLD activities of MDCK-D1 cells, distinct from that present in nuclei, might be regulated by ARF, especially since MDCK cells, as well as other cell types, demonstrate multiple PLD activities(35, 36, 37) .
Collectively, the
present results, along with our previous data(18) , indicate
that nuclear PLD may be regulated via multiple cellular constituents,
acting in sequence. The data strongly suggest that a PKC-driven
phosphorylation reaction is required for the subsequent ability of RhoA
to activate nuclear PLD. PA, the primary product of PLD action on
phospholipids, has been shown to be mitogenic for certain cell
types(8) . An intriguing but speculative idea is that products
of nuclear PLD may help regulate binding of certain PKC isotypes to the
nuclear membrane. In this regard, PA has been observed to specifically
bind and activate PKC-, a PKC isotype suggested to play a key role
in mitogenesis and a number of nuclear
events(38, 39) . This raises the possibility that PA
generated by PLD in the nucleus increases PKC-
binding and
function in this cellular compartment. In addition, it is also possible
that activation of nuclear PLD alters lipid composition and
lipid-dependent functional activities in the nucleus.