From the
Many receptors, in response to their specific ligands, trigger
activation of phospholipase D (PLD), resulting in the production of
phosphatidic acid which, in turn, is acted upon by a specific
phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol.
We report here that isolated nuclei from Madin-Darby canine kidneys
(MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence
of ATP. PLD activity was measured in the presence of ethanol, by
quantitating the production of phosphatidylethanol. Non-phosphorylating
ATP analogs were unable to substitute for ATP in activating PLD,
indicating that ATP acts as a phosphoryl group donor in a
kinase-mediated phosphorylation reaction. The protein kinase C
inhibitors chelerythrine and calphostin completely suppressed the
ATP-induced nuclear PLD, implicating protein kinase C as the kinase
involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In
the absence of ethanol, phosphatidic acid was detected in ATP-treated
nuclei. Accumulation of phosphatidic acid preceded or closely
paralleled that of diacylglycerol, suggesting a precursor-product
relationship. Consistent with those results, we detected phosphatidate
phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of
phosphatidic acid and diacylglycerol levels at increasing amounts of
ethanol demonstrated that PLD and phosphatidate phosphohydrolase are
responsible for generating the majority of the diacylglycerol
accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate
diacylglycerol from the concerted action of those two enzymes provides
a means to regulate nuclear lipid synthesis as well as protein kinase C
activity.
Signal transduction processes are often initiated by the
hydrolysis of phospholipids catalyzed by phospholipases at the plasma
membrane generating lipid second messengers
(1) . Activation of
phospholipase D (PLD)
Recovery of marker enzyme activities in nuclear fractions was
substantially less than was observed for PLD activity (12.0 ±
1.5% of the total cellular activity), assessed using
[
Phospholipids comprised 63 ± 4% of the
It has recently been suggested that ATP
potentiates PLD activity in permeabilized cells
(17, 18) because it is required for phosphoinositide kinase to
synthesize phosphatidylinositol 4,5-bisphosphate, a phospholipid that
has been demonstrated to increase PLD activity in cell-free
systems
(17, 19) . However, the fact that the specific
PKC inhibitors calphostin and chelerythrine completely suppress the
ATP-dependent PLD activity of preparations of MDCK-D1 nuclei argues
strongly against the possibility that increase in synthesis of
phosphatidylinositol 4,5-bisphosphate is the mechanism whereby ATP
promotes PLD activation in MDCK-D1 cells. Instead, our data support a
model for nuclear PLD activation whereby the major ATP-requiring step
is nuclear-associated PKC.
In the absence of ethanol, PA was
detected in ATP-treated nuclei with a time dependence of accumulation
similar to that seen for PEt (Fig. 3A). Accumulation of
PA preceded or closely paralleled that of DAG, suggesting a
precursor-product relationship. This could occur if MDCK-D1 cell nuclei
contained a PA phosphohydrolase, the enzyme activity that converts PA
into DAG. Aliquots of the nuclear fraction were assayed for their
ability to convert exogenous PA into DAG. Fig. 3(B and
C) shows that this conversion occurred in a time- and protein
concentration-dependent manner, demonstrating association of PA
phosphohydrolase with MDCK-D1 cell nuclei.
(
)
results in the production
of phosphatidic acid (PA), which in turn is acted upon by PA
phosphohydrolase to produce diacylglycerol (DAG)
(1, 2) .
Both of these products can serve second messenger functions and the
sustained activation of PLD is believed to be the major route for
generation of these two lipid messengers in many
cells
(1, 2, 3, 4, 5) . DAG
through its activation of protein kinase C (PKC) regulates functions in
the cell nucleus, in which PKC mediates phosphorylation of regulatory
and structural proteins. Moreover, nuclear accumulation of DAG has been
observed in a variety of cell systems
(6, 7) . In
addition, PA and its lyso-derivative lysoPA modulate nuclear events,
including DNA synthesis (8). It is unclear whether PKC-mediated events
in the nucleus result from generation of DAG at the plasma membrane. In
the current work, we have tested and confirmed the hypothesis that cell
nuclei possess the ability to generate DAG and PA through a PLD and PA
phosphohydrolase pathway. These two enzymes, working in concert, are
responsible for generating the majority of the nuclear DAG production
and therefore probably contribute to regulation of nuclear PKC.
Materials
[H]Palmitic acid
(specific activity 54 Ci/mmol) and
1,2-dipalmitoyl-[U-
C]glycero-3-phosphate
(specific activity 144 mCi/mmol) were obtained from DuPont NEN. Phorbol
12-myristate 13-acetate (PMA), ATP, AMP-PNP, AMP-PCP, herbimycin, and
H89 were from Sigma. Chelerythrine was obtained from LC Services
(Woburn, MA). Calphostin was from Calbiochem. G-60 thin layer
chromatography plates were obtained from either Whatman or Analtech
(Newark, DE). The organic solvents were from Fisher.
Growth and Treatments of MDCK-D1 Cells
MDCK-D1
cells were grown as described elsewhere
(9) . The cells were
labeled the day before confluence with
[H]palmitic acid (3 µCi/ml) in
Dulbecco's modified Eagle's medium. After removal of
labeling medium, cultures were equilibrated with serum-free medium
containing 1 mg/ml bovine serum albumin at 37 °C for 45 min. Cells
were then incubated at 37 °C with PMA (80 nM) or ATP (300
µM) for 30 min in the presence of 1% ethanol. Reactions
were stopped by aspirating the media and adding cold hypotonic buffer
(see below). Nuclei were separated at 4 °C as described below, and
nuclear PEt content was determined by thin-layer chromatography after
extraction of total nuclear lipids
(9) .
Preparation of Nuclei from MDCK-D1 Cells
The
method of York and Majerus
(10) was used with slight
modification to obtain purified nuclei from MDCK-D1 cells. Cells were
labeled with 3 µCi/ml [H]palmitic acid for 20
h. After this time, the cells were washed twice with cold
phosphate-buffered saline, overlaid with a hypotonic buffer consisting
of 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 10 µM benzamidine, and 10
µM aprotinin (buffer A), and scraped from the plate. Cells
were subjected to 15 passes in a Potter-type Teflon-on-glass
homogenizer and spun at 500
g for 5 min. The
supernatant was discarded, and the pellet was resuspended in buffer A.
The resulting nuclear suspension was layered onto a 200-µl sucrose
cushion (50% (w/v)) in buffer A and spun at 15,000
g for 1 min in an Eppendorf centrifuge. The nuclei pelleted through
the cushion were resuspended in buffer A. Purity of these nuclei was
assessed by electron microscopy and by measuring enzyme marker
activities. These nuclei contained less than 7% of the total cellular
endoplasmic reticulum enzyme NADPH cytochrome c reductase,
less than 4% of the total cellular cytosolic marker lactate
dehydrogenase, and less than 1% and 3% of the total cellular light
membrane (including plasma membrane) markers 5`-nucleotidase and
alkaline phosphatase, respectively. All of these enzyme markers were
assayed as described elsewhere
(11) . Enzyme marker activities
were also assayed in nuclear preparations from activated cells (80
nM PMA, or 300 µM ATP for 30 min), and the values
obtained were similar to those observed for nuclei from unstimulated
cells.
C]phosphatidylcholine as exogenous substrate
under the conditions described by Huang et al.(12) .
This indicates that nuclear-associated PLD activity does not appear to
arise from contamination from other cellular compartments.
H
radioactivity present in nuclear lipids, the remaining being
incorporated into neutral lipids.
H radioactivity in
phospholipids was distributed among major phospholipid classes as
follows: phosphatidylcholine, 37 ± 1%; phosphatidylethanolamine,
18 ± 1%; phosphatidylinositol/phosphatidylserine, 5 ± 1.
Phospholipase D Activity Assay
Nuclear fractions
(up to 50 µg of protein) were incubated in Olson's buffer (25
mM Hepes, 100 mM KCl, 3 mM NaCl, 5
mM MgCl, 1 µM CaCl
, 1
mM phenylmethylsulfonyl fluoride, 10 µM
benzamidine, 10 µM aprotinin, pH 7.4)
(13) at 37
°C along with the indicated ATP concentration and 1.5% ethanol (285
mM). Final volume was 200 µl. Total lipids were extracted
as described previously
(14) , and PEt was resolved by thin layer
chromatography on Silica Gel G plates using the upper phase of a system
consisting of ethyl acetate/isooctane/acetic acid/water (130:20:30:100,
by volume) (9). When inhibitors were used, they were added at the
indicated concentrations for 30 min prior to and during the
incubations. PEt production is expressed as the percentage of
radioactivity in PEt compared with the total radioactivity in nuclear
phospholipids.
PA Phosphohydrolase Activity Assay
PA
phosphohydrolase activity was determined as described by Day and Yeaman
(15) with slight modification. The substrate
[C]glycerol-labeled PA was delivered as mixed
micelles with Triton X-100 at a detergent/phospholipid mole ratio of
10:1. Assays were conducted at 37 °C. The incubation mixture
contained in a final volume of 0.1 ml: 100 µM
[
C]PA substrate (0.025 µCi/assay), 1
mM Triton X-100, 50 mM Tris-HCl (pH 7.1), 10
mM
-mercaptoethanol, 2 mM MgCl
, 1
mM EDTA, 1 mM EGTA, and the indicated amount of
nuclear protein. After the indicated times the reaction was stopped and
[
C]PA and [
C]DAG were
separated by thin layer chromatography as described
elsewhere
(13) .
RESULTS AND DISCUSSION
The widely used method for detection of PLD is based on the
formation of PEt, a product that is generated from PLD by a
transphosphatidylation reaction when ethanol is present
(16) . By
measuring PEt, we have recently characterized the activation of PLD by
P purinergic receptors and by phorbol ester in MDCK-D1
cells
(9) . In the course of assessing the intracellular
distribution of PEt generated upon stimulation of MDCK-D1 cells with
PMA or ATP, we have found accumulation of PEt in isolated nuclei from
activated cells, but not in nuclei from unstimulated cells
(Fig. 1). Cellular stimulation by phorbol ester and ATP caused
respectively a 50- and 16-fold increase in nuclear PEt levels as
compared to unstimulated cells. Nuclear PEt production represented 19
± 5% and 17 ± 4% of total cellular PEt production in
response to PMA and ATP, respectively.
Figure 1:
Accumulation of PEt in isolated nuclei
from MDCK-D1 cells. Intact MDCK-D1 cells were stimulated with ATP (300
µM) (graybar), PMA (80 nM)
(blackbar), or neither (whitebar)
for 30 min in the presence of 1% ethanol. After extraction, PEt was
separated by thin-layer chromatography. Results are shown as means
± S.E. from three different experiments with triplicate
determinations and are expressed as a percentage of radioactivity in
PEt relative to total radioactivity in nuclear
phospholipids.
Inasmuch as PEt appears to be
a metabolically inert phospholipid and it is the exclusive product of a
PLD activity, its accumulation in a given compartment would suggest the
presence of a PLD in such a compartment. Therefore, preparations of
isolated nuclei were tested for PLD activity. Nuclei were isolated
without using detergents, in order to achieve nuclear envelope
integrity, which, in turn, would serve as a source for labeled
phospholipid substrate in our assay. When nuclei from
[H]palmitate-labeled cells were incubated at 37
°C in the presence of 1.5% ethanol, a product co-migrating with
authentic PEt was formed (Fig. 2A). These results
demonstrate the presence of a PLD activity associated with the isolated
nuclei. Importantly, substantial PEt production could only be measured
if ATP was present in the incubation medium. ATP promoted accumulation
of nuclear PEt in a time- and concentration-dependent manner
(Fig. 2, B and C). The non-phosphorylating
adenine trinucleotides AMP-PNP and AMP-PCP were unable to substitute
for ATP in activating PLD (Fig. 2D), thus strongly
suggesting that ATP acts as a phosphoryl group donor in a
kinase-mediated phosphorylation reaction.
Figure 2:
ATP-regulated PEt production in nuclei
from MDCK-D1 cells. A, autoradiography of phospholipids from
MDCK-D1 cell nuclei after TLC separation. B, time course of
PEt accumulation in the presence () or absence (
) of 500
µM ATP. C, concentration response of the ATP
effect measured at 30 min. D, effect of non-hydrolyzable ATP
analogs on PEt production. Effect of chelerythrine (E) or
calphostin (F) on PEt production in the absence (
) or
presence (
) of 500 µM ATP. Results shown 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.
In searching for the
kinase involved in ATP-dependent PLD activation in MDCK-D1 nuclei, we
employed a number of well established kinase inhibitors, including
chelerythrine and calphostin, herbimycin, and H89, selective inhibitors
of PKC, protein tyrosine kinase, and cAMP-dependent protein kinase,
respectively. The two PKC inhibitors, chelerythrine and calphostin,
were able to completely suppress the ATP-induced nuclear PLD
(Fig. 2, E and F), whereas herbimycin and H89
were ineffective (data not shown). These results implicate PKC as the
kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1
cells. In keeping with this finding, Western blot analyses of nuclear
preparations revealed the presence of the PKC isoforms ,
,
and
associated with nuclei from MDCK-D1 cells (data not shown).
PKC
has been demonstrated to play a critical role in phorbol
ester- and receptor-promoted activation of PLD in MDCK-D1
cells
(9) . Further proof for the involvement of PKC in
regulating nuclear PLD was obtained by directly adding PMA to the
nuclear preparations. Inclusion of PMA (80-250 nM)
increased by 1.6 ± 0.2-fold the ATP-activated PLD activity of
MDCK-D1 cell nuclei.
Figure 3:
Dynamics of PA and DAG production in
nuclei. A, time course of PA () and DAG (
)
accumulation in ATP-treated nuclei. B, effect of protein
concentration (10-min incubation), and C, effect of time (10
µg of protein), on PA phosphohydrolase activity in nuclei. Results
are given as means ± S.E. from triplicate determinations in
single representative experiments. Each set of experiments was repeated
at least three times with similar results.
In order to quantitate
how much of the nuclear DAG accumulation was derived directly from the
PA produced by the ATP-activated PLD, measurements were conducted in
the presence of increasing amounts of ethanol, since ethanol diverts
PLD activity from PA formation. Consequently, in the presence of
ethanol, DAG levels derived from PLD action should decrease. As shown
in Fig. 4, increasing concentrations of ethanol increased
formation of PEt in parallel with decreases in PA and DAG. At
saturating concentrations of ethanol, at which PEt formation reaches a
plateau, DAG production was completely abolished. This result is
consistent with the conclusion that the majority of DAG accumulating in
nuclei derives from the ATP-regulated PLD activity.
Figure 4:
Accumulation of PEt (), PA
(
), and DAG (
) in the presence of increasing ethanol
concentrations. Nuclei from [
H]palmitic
acid-labeled MDCK-D1 cells were incubated as described under
``Experimental Procedures'' in the presence of the indicated
ethanol concentrations. Results are given as means ± S.E. from
triplicate determinations in a single experiment, which is
representative of three different ones.
In recent years,
increasing evidence has accumulated to indicate that nuclei have very
active lipid metabolism which may play a crucial role in nuclear
function (reviewed in Ref. 20). A nuclear phosphoinositide cycle
entirely separate from that operating in the plasma membrane has been
recently elucidated
(21, 22) . Activation of this cycle
by plasma membrane receptors leads to transient increases in nuclear
inositide-derived DAG
(21, 22) . Our data suggest an
alternative and novel mechanism for increases in nuclear DAG: the
sequential actions of PLD and PA phosphohydrolase endogenous to or
closely associated with the cell nucleus. The ability of PKC to
activate nuclear PLD suggests the existence of a signaling pathway
whereby plasma membrane receptors activate one or more forms of PLC (or
PLD) thereby promoting activation of PKC, and PKC, in turn, promoting
activation of nuclear-associated PLD. This novel signaling pathway
located in the nucleus appears to be responsible for the major portion
of the DAG generated by nuclei. This DAG could be utilized both for
activation of PKC as well as for synthesis of lipids, assuming
accessibility to other enzymes required for lipid synthesis
,
-imino)triphosphate; AMP-PCP, adenosine
5`-O-(
,
-methylene)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.