(Received for publication, August 29, 1996, and in revised form, November 5, 1996)
From the Two forms of phospholipase D (PLD) have been
found to be present in nuclei isolated from rat hepatocytes by
measuring phosphatidylbutanol produced from exogenous radiolabeled
phosphatidylcholine in the presence of butanol. In nuclear lysates from
either rat liver or ascites hepatoma AH 7974 cells, the PLD activity
was markedly stimulated by a recombinant ADP-ribosylation factor (rARF)
in the presence of the guanosine 5 It has been known that cell nuclei contain a variety of enzymes
generating lipid second messengers, such as sphingomyelinase (1),
phospholipase A2 (2), PI1-specific
phospholipase C (3-6) and lipid kinases (7). Growth factors seem to be
able to affect the phosphoinositide metabolism in nuclei, suggesting a
subtle regulation during cell activation (8, 9). In addition,
accumulation of diacylglycerol (DG) in nuclei and translocation of
protein kinase C to them have been demonstrated in a variety of cell
types (10, 11). A large rise in mass of DG, with only small changes in
mass of phosphoinositides, suggested a source of DG other than
polyphosphoinositides, for example, phosphatidylcholine (PC), in nuclei
(12). A recent study (13) has demonstrated that phosphatidylethanol
formation from phosphatidylcholine, which was specifically catalyzed by phospholipase D (PLD) in the presence of ethanol, was induced by PMA in
nuclei isolated from kidney cells and that nuclei possess the ability
to generate DG and phosphatidic acid through the PLD:phosphatidic acid
phosphohydrolase pathway. Thus, upon cell stimulation with agonists,
the enzymes involving PC metabolism are considered to be activated in
the nucleus as well as in the plasma membrane.
It was demonstrated that PLD activity can be regulated by several
factors (14, 15); oleate, small G proteins (ARF and Rho family),
phosphatidylinositol 4,5-bisphosphate (PIP2),
phosphatidylethanolamine (PE), Ca2+, protein kinase C,
protein tyrosine kinase. Recently, two different types of PLD,
oleate-dependent and ARF-dependent, were
isolated from rat brain membranes (16). The
oleate-dependent PLD was purified from pig lung microsomes
(17), and ARF-dependent PLD activity was found to be
abundant in Golgi-enriched membranes from several cell lines (18). More
recently, a gene of human ARF-dependent PLD
(hPLD1) has been cloned (19). It has been shown that the
membrane-associated PLD activity was modulated by protein kinase C and
Rho family (20-24). These observations suggest the presence of
different isoforms of PLD.
The nuclear PLD activity from Madin-Darby canin kidney (MDCK)-D1 cells
was observed to be regulated by protein kinase C and RhoA (23). It was
also shown that an oleate-dependent form of PLD was present
in rat brain neuronal nuclei and inhibited by acidic phospholipids,
such as PIP2 (25). However, the physiological roles of PLD
in nuclei remain unclear.
In this study, we have demonstrated that two forms,
ARF-dependent and oleate-dependent PLD
activities, were present in the isolated nuclei from rat resting liver,
regenerating liver, and rat ascites hepatoma cells AH7974 and
characterized their PLD activities using exogenous radiolabeled PC
substrates. Furthermore, we have shown a transient elevation of the
nuclear ARF-dependent PLD activity, but not the
oleate-dependent PLD activity during S-phase of liver
regeneration.
[2-palmitoyl-9,10-3H]Dipalmitoylphosphatidylcholine
(DPPC) (37.5 Ci/m mol) was obtained from DuPont NEN. Egg
phosphatidylcholine (egg PC), PIP2, PE, and sodium oleate
were from Sigma. GTP Nuclei were isolated and purified from
rat ascites hepatoma AH7974 cells and rat liver cells as described
previously (2, 26). They were highly pure as examined by electron
microscopic examination and by determining marker enzymes for
microsomes (glucose-6-phosphatase), plasma membrane (5 Rats (7-week-old) of inbred
Donryu strain were used in all experiments. Partial hepatectomy was
performed under light ether narcosis, and two-thirds of the liver was
surgically removed according to the method of Higgins and Anderson
(28). Livers were obtained as follows: control livers from nontreated
and sham-operated rats and regenerating livers from partially
hepatectomized rats (1, 2, 5, 8, 12, 18, 20, 22, 24, 26, 28, 30, and
32 h post-hepatectomy).
The nuclear fractions
were precipitated by centrifugation at 10,000 × g for
5 min and resuspended in lysis buffer containing 25 mM
HEPES/NaOH buffer, pH 7.4, 100 mM KCl, 5 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, and
10 mg/ml leupeptin. The suspended nuclei were sonicated three times
15 s each. The PLD activities were assayed by slight modification
of the procedure of Massenburg et al. (16). For the assay of
oleate-dependent PLD activity, egg PC vesicles (20 µl)
containing 15 nmol of egg PC and 400,000 dpm of [3H]DPPC
were prepared by sonication and then mixed with 5 µl of 12 mM sodium oleate. The substrates were added to nuclear
lysates in a total volume of 120 µl containing 50 mM
HEPES/NaOH buffer, pH 7.0, 2 mM EGTA, 500 mM
KCl, 1 mM MgCl2, 2.1 mM
CaCl2, and 0.3% (v/v) butanol and then incubated at
30 °C for 30 min.
For the assay of ARF-dependent PLD activity, mixed lipid
vesicles (PE/PIP2/egg PC, 160/14/10 µM)
containing [3H]DPPC to yield 400,000 dpm/assay were
prepared. The substrates (25 µl) were added to 10 ml of nuclear
lysates in a total of 120 µl containing 50 mM HEPES/NaOH
buffer, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 1.2 mM CaCl2, 5 µM recombinant ARF
(rARF), 30 µM GTP Reactions were terminated by addition of 0.8 ml of chloroform/methanol
(1:2, v/v) and 0.4 ml of chloroform and 0.3 ml of 200 mM
KCl, 5 mM EDTA. Lipids were extracted by the method of
Bligh and Dyer (29) and separated on TLC plates (Silica gel 60A) in a
solvent system using the upper phase of ethyl
acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10, v/v) as
described by Chalifa et al. (30). The plates were exposed to
I2 vapor and [3H]PBut formation was
identified by comigration with the authentic standard. Silica gels
scraped off from the plates were mixed with scintillation mixture, and
radioactivities were counted in a liquid scintillation counter.
Protein
concentration was determined using a Bio-Rad Bradford assay reagent
using bovine serum albumin as standard. The isolated nuclei were
resuspended in the sample buffer (60 mM Tris, 10% glycerol, 45 mM mercaptoethanol, 80 mM sodium
dodecyl sulfate, pH 6.8) and boiled for 3 min. Proteins were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 13 and 8% polyacrylamide gels and transferred to the polyvinylidene
difluoride membrane and then incubated with antibodies.
Antigen-antibody complexes were detected by the chemiluminescence (ECL)
method (Amersham Corp.). The intensities of bands were quantitated by densitometry (ATTO Densitograph AE-6900M).
Escherichia coli bearing ARF1 plasmid was
gift from Dr. Joel Moss and recombinant ARF1 was purified by
chromatography on DEAE-Sephacel and Sephacryl S-300 columns as
described (31). Protein preparation showed only one band on
Coomassie-stained SDS-polyacrylamide gels.
It has been demonstrated that two types of
PLD activity, oleate-dependent and
ARF-dependent, have been reported in mammalian cells and
tissues (14, 16, 17). PLD activity was examined in nuclei isolated from
rat resting liver and AH7974 cells using the exogenous radiolabeled PC
substrates in the presence or absence of rARF (5 µM) and
GTP
Nuclear ARF-dependent and oleate-dependent PLD
activities in liver cells and AH cells
The nuclear PLD activities of both rat resting liver and AH7974 cells
were increased by rARF in the presence of 30 µM GTP
It has been known that PKC had a synergistic stimulatory effect of the
ARF-dependent PLD activity (32). To examine the effect of
PKC on the nuclear ARF-dependent PLD activity, the nuclear lysates of the rat resting liver and AH7974 cells were incubated with
GTP
To examine the oleate-dependent PLD activity, the rat liver
and AH7974 nuclear were incubated with [3H]DPPC in egg PC
vesicles as substrate in the presence of 0.5 mM oleate. The
rate of [3H]PBut formation of their nuclear lysates was
linear for at least 30 min, and a linear increase of
[3H]PBut formation was observed up to 50 µg protein of
their nuclear lysates (data not shown). Characterization of the
oleate-dependent PLD activity of the AH7974 nuclei was
shown in Fig. 4. An optimal pH was 6.0-7.0 in 50 mM HEPES/NaOH buffer (Fig. 4A). Lower
concentration of Ca2+ less than 0.1 mM had
little effect on the [3H]PBut formation induced by
oleate, but its higher concentrations over 0.5 mM were
rather inhibitory. On the other hand, Mg2+ stimulated the
PLD activity with the maximal level at 1 mM (Fig. 4B). Oleic acid was the most potent in stimulating the
AH7974 nuclear PLD activity. The [3H]PBut formation was
increased by oleate in a dose-dependent manner with a
maximal level at 0.5 mM (Fig. 4C). The optimal
molar ratio of sodium oleate to egg PC was approximately 4. Other
unsaturated fatty acids, linoleic and arachidonic acids, at the same
concentration enhanced the PLD activity to lower extents, as compared
with oleate (Fig. 4D). In contrast, saturated fatty acids,
palmitic and stearic acids, had no effect. The biochemical properties
of the oleate-dependent PLD activity in rat liver nuclei
were similar to those of AH7974 nuclei (data not shown). The nuclear
oleate-dependent PLD activity was not affected by GTP
In order to examine whether the nuclear PLD activity is
somehow associated with cell proliferation, PLD activity was measured in nuclei isolated from regenerating liver of partially hepatectomized rats. The ARF-dependent PLD activity began to increase
23 h after hepatectomy, reaching a peak at 26-28 h (Fig.
5B). At 28 h after hepatectomy the
nuclear ARF-dependent PLD activity was approximately 5-fold
higher than that of the control liver nuclei. On the other hand, the
oleate-dependent PLD activity remained unchanged during liver regeneration (Fig. 5C). The GTP
-It has been reported that cell stimulation with various
agonists induces translocation of protein kinases to nuclei as a result
of DG formation by PI-PLC activation (10, 11). To see the proteins
translocated to the nuclei after partial hepatectomy, the nuclei from
S-phase liver cells (28 h after partial hepatectomy) were examined by
Western blotting with various antibodies. As shown in Fig.
6A, the levels of PKC
Time-dependent changes in PKC isozyme levels in nuclei
during liver regeneration showed different profiles among PKC isozymes (Fig. 6B). There was a remarkable elevation of PKC To examine the intranuclear G protein levels, the isolated nuclei from
AH7974, the resting rat liver, and regenerating liver (28 h after
partial hepatectomy) were blotted with various specific antibodies.
Analysis of the Western blotting revealed the presence of low molecular
weight GTP-binding proteins, ARF, RhoA, and Cdc42 and heterotrimeric
GTP-binding protein, Gi2 (Fig. 7). The nuclear ARF and
RhoA levels of AH7974 cell were 9- and 2-fold higher than those of rat
resting liver cells, respectively, although the Cdc42 and Gi2 levels
were almost similar between both nuclei. ARF protein was scant in the
rat resting liver nuclei, but it was 5-fold increased in the nuclei of
S-phase. A little increase of nuclear RhoA level was observed in the
S-phase (1.3-fold).
We have demonstrated here that two forms of PLD activity,
GTP Several studies have shown that ARF plays an essential role in membrane
trafficking by controlling the reversible assembly of the coat protein
complex on the surface of Golgi membranes (40) and that ARF and
ARF-stimulated PLD activity is enriched in Golgi membranes (18, 41). On
the other hand, Boman et al. (42) have suggested a role for
ARF in nuclear vesicle dynamics during mitosis. Our results indicated
that the specific activity of the nuclear ARF-dependent PLD
activity, but not oleate-dependent enzyme, was transiently
elevated in S-phase of rat regenerating liver cells. Moreover, the
ARF-dependent PLD activity was extremely high in the nuclei
of rat hepatoma AH7974 cells compared with that of the resting liver
cells. The nuclear ARF levels was high in the AH7974 cells, and the
increase of nuclear ARF level was observed in S-phase. Thus, one would
speculate that the nuclear ARF-dependent PLD may be
activated during S-phase to generate nuclear signaling molecules
required for DNA synthesis or to enter into G2 phase.
There are some possible explanations for the transient elevation of the
nuclear ARF-dependent PLD activity during S-phase of rat
liver regeneration. First, the GTP A number of studies have provided evidence suggesting roles for the
activation of nuclear PKC in nuclear functions involving phosphorylation of transcription factors and DNA replication factors (46). Also it has been shown that the activation of nuclear PKC is
correlated with an increase in nuclear DG. Divecha et al. (8, 10) have demonstrated that a turnover of nuclear PI is important in
modulation of nuclear PKC in NIH3T3 cells. Furthermore, these notions
are supported by the presence of nuclear PLC (3-6). On the other hand,
Jarpe et al. (12) have shown that the source of the
thrombin-induced nuclear DG in fibroblasts is PC, not PI by examining
the molecular species profiles. These reports suggest that both PI and
PC contribute to the formation of nuclear DG, but the hydrolyzing
enzymes are differently located in the nucleus; PLC is associated with
the internal matrix (7), while the PC-hydrolyzing enzyme exists in the
nuclear envelope (11). The result was supported by our results; the
envelope-depleted nuclei from rat liver cells and AH7974 cells after
treatment with Triton X-100 as described by Payrastre (7) had lesser
activity of the nuclear ARF-dependent PLD, suggesting that
the ARF-dependent PLD activity was located in the nuclear
envelope (data not shown).
Many laboratories have demonstrated the nuclear localization of PKC
isozymes in both resting and stimulated cells (11, 47-49). PKC We thank Dr. Joel Moss (National Institutes
of Health) for kind gifts of the ARF1 plasmid and the anti-ARF antibody
and Dr. Alan Hall (University College London) for generous gift of the C3 exoenzyme plasmid.
Department of Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-O-(3-thiotriphosphate)
(GTP
S) and phosphatidylinositol 4,5-bisphosphate. ATP and
phorbol-12-myristate 13-acetate had no synergistic effect on this PLD
activity. On the other hand, the nuclear PLD was stimulated by
unsaturated fatty acids, especially by oleic acid. The
ARF-dependent nuclear PLD activity was increased in the
S-phase of the regenerating rat liver after partial hepatectomy and
also was much higher in AH 7974 cells than in the resting rat liver. In
contrast, the levels of the oleate-dependent PLD activity
remained constant throughout the cell cycle in liver
regeneration. The intranuclear levels of the stimulating proteins of
the nuclear PLD activity, e.g. ARF, RhoA, and protein
kinase C
increased in the S-phase of the regenerating liver. These
results suggested that the nuclear ARF-dependent PLD activity may be
associated with cell proliferation.
Materials
S was from Boehringer
(Mannheim, Germany). Antibody against ARF1 was a generous gift from Dr.
Joel Moss (National Institutes of Health, Bethesda, MD). Antibodies to
PKC isozymes (
,
I,
II,
,
,
), RhoA, Cdc42, Rac1,
ERK-2, and P38 MAP kinase were purchased from Santa Cluz Biotechnology
(San Francisco, CA). Anti-Gi2
antibody was from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan).
-nucleotidase),
and mitochondoria (cytochrome oxidase). DNA polymerase
activity was
measured in isolated nuclei as described previously (27). The isolated
nuclei were resuspended in 0.25 M sucrose, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM phenylmethylsulfonyl fluoride.
S, and 0.3% butanol and incubated at
37 °C for 1 h.
Characterization of PLD Activity in Rat Liver and Rat Ascites
Hepatoma Cell Nuclei
S (30 µM) or oleate (0.5 mM). As shown in Table I, the specific activities of the
ARF-dependent and oleate-dependent PLD of
AH7974 nuclei were approximately 10- and 3-fold higher than those of
rat resting liver nuclei, respectively. When used
PE/PIP2/PC vesicles as substrate, the PLD activity without stimulators was high in the AH7974 nuclei. In the AH7974 nuclei, as
shown in Fig. 1, the [3H]PBut formation in
the absence of GTP
S showed a linear increase in a
time-dependent manner, although a much less increase than that with GTP
S, indicating the presence of a GTP
S-independent PLD
activity. Addition of GTP
S (30 µM) to AH7974 nuclear
lysate increased the PLD activity in a time-dependent
manner (Fig. 1A). The rate of [3H]PBut
formation was linear for at least 60 min. The PLD activity without
GTP
S was not affected by ATP (0.5 mM). The PLD
activities with or without GTP
S were increased by PIP2
in a dose-dependent manner (Fig. 1B). The ratio
of PIP2:PC was 3:1 for the maximal activation. The PLD
activity without GTP
S in the nuclei isolated from rat resting liver
was very low, even in the presence of PIP2.
S (30 µM) and [3H]DPPC in egg PC vesicles (substrate
2) with 0.5 mM oleate as described under "Experimental
Procedures." Results are given as mean ± S.E. from duplicate
determinations of three different experiments.
Rat liver cells
AH cells
nmol/h/mg protein
None (substrate
1)
0.19 ± 0.08
0.85 ± 0.13
ARF/GTP
S
0.61 ± 0.12
6.02 ± 0.67
None
(substrate 2)
0.08 ± 0.01
0.08 ± 0.02
Oleate
0.49 ± 0.09
1.52 ± 0.23
Fig. 1.
Effects of incubation time (A)
and PIP2 (B) on the nuclear PLD activity.
A, the AH nuclear lysates (2 µg of protein) were incubated
with or without 30 µM GTPS or 0.5 mM ATP
without GTP
S for indicated times. [3H]PBut formation
was measured by using [3H]DPPC in PE/PIP2/egg
PC vesicles as substrate as described under "Experimental
Procedures." B, [3H]PBut formation was
measured by using [3H]DPPC in PE/PIP2/egg PC
(indicated various molar ratios of PIP2/egg PC) vesicles as
substrate. Results are given as mean ± S.E. from duplicate
determinations of three different experiments.
[View Larger Version of this Image (28K GIF file)]
S in a dose-dependent manner, reaching a maximal level at 8 µM (Fig. 2A). Linear increases
in the ARF-dependent PLD activity were observed up to 2 and
5 µg of proteins of the AH7974 and rat resting liver nuclear lysates,
respectively (Fig. 2B). Both nuclear
ARF-dependent PLD activities were limitedly stimulated by
low concentration of Ca2+ with maximal effect at 0.3 µM and inhibited by higher concentrations than 1 µM (Fig. 2C). Mg2+ stimulated both
nuclear ARF-dependent PLD activities with maximal concentration of 3 mM (Fig. 2D).
Fig. 2.
Effects of ARF (A), protein
concentration (B), Ca2+
(C), and Mg2+ (D) on the
nuclear PLD activity. The nuclear lysates from rat liver () and
AH cells (
) were incubated with 30 µM GTP
S and
indicated concentrations of rARF (A); GTP
S (30 µM), rARF (5 µM), and indicated
concentrations of proteins (B); GTP
S (30 µM), rARF (5 µM), and indicated
concentrations of Ca2+ (C); and GTP
S (30 µM), rARF (5 µM), and indicated
concentrations of Mg2+ (D).
[3H]PBut formation was measured by using
[3H]DPPC in PE/PIP2/egg PC vesicles as
substrate as described under "Experimental Procedures." Results are
given as mean ± S.E. from duplicate determinations from three
different experiments.
[View Larger Version of this Image (31K GIF file)]
S (30 µM), rARF (5 µM), PMA (100 nM), and ATP (0.5 mM) or their combination. As
shown in Fig. 3, rARF was most potent activator for both
nuclear PLD activities. PMA had no synergistic stimulatory effect on
the nuclear ARF-dependent PLD activity in both nuclei.
Fig. 3.
Effects of ARF and PMA on the nuclear PLD
activity. The nuclear lysates (3 µg of protein) from resting rat
liver and AH cells (2 µg of protein) were incubated with or without
GTPS (30 µM), rARF (10 µM), PMA (100 nM), or combination of rARF (10 µM)/GTP
S
(30 µM) and PMA (100 nM)/rARF (10 µM)/GTP
S (30 µM) for 1 h.
[3H]PBut formation was measured with
[3H]DPPC in PE/PIP2/egg PC vesicles as
substrate as described under "Experimental Procedures." Results are
given as mean ± S.E. from duplicate determinations from three
different experiments.
[View Larger Version of this Image (40K GIF file)]
S,
PIP2, rARF, or PMA (data not shown), indicating that the
nuclear oleate-dependent PLD is distinct from the
ARF-dependent one.
Fig. 4.
Effects of pH (A), divalent
cations (B), oleate (C), and other fatty acids
(D) on the nuclear PLD activity. The AH nuclear
lysates (10 µg of protein) were incubated with various stimulants for
30 min. The [3H]PBut formation was measured by using
[3H]DPPC in egg PC vesicles as substrate in the presence
of 0.5 mM oleate (A and B), 0.5 mM various sodium fatty acids: PAL, palmitic; STE, stealic; OLE, oleic; LIN,
linoleic; ARA, arachidonic fatty acid (D) or the
indicated concentrations of oleate (C). Effect of pH
(A) was examined with acetate buffer (pH 4-6) and
HEPES/NaOH buffer (pH 7-9). Results are given as mean ± S.E.
from duplicate determinations of three different experiments.
[View Larger Version of this Image (36K GIF file)]
S-independent PLD
activity was very low in the regenerating liver nuclei and remained
unchanged (Fig. 5A). The transient increase in the
ARF-dependent PLD activity was observed 2 ~ 3 h
later following the increase in the DNA polymerase
activity (Fig.
5D), which is a marker for S-phase (3, 27), suggesting that
the ARF-dependent PLD is induced along progression of DNA
synthesis in S-phase (33).
Fig. 5.
Changes in the nuclear PLD and DNA polymerase
activities during liver regeneration. Livers were obtained
from nontreated, sham-operated rats, and regenerating livers from
partially hepatectomized rats (1, 2, 5, 8, 12, 18, 20, 22, 24, 26, 28,
30, and 32 h post-hepatectomy). Nuclei were prepared from each
liver as described under "Experimental Procedures."
[3H]PBut formation was measured in nuclear lysates (3 µg of protein) of regenerating livers from operated rats (
) and of
control livers from sham operated rats (
) by using
[3H]DPPC in PE/PIP2/egg PC vesicles for the
ARF-dependent PLD activity (A, B) and
[3H]DPPC in egg PC vesicles for the
oleate-dependent PLD activity (C) as described
under "Experimental Procedures." DNA polymerase
activity was
determined by the method described previously (27) (D).
Results are given as mean ± S.E. from duplicate experiments of
three different experiments.
[View Larger Version of this Image (25K GIF file)]
, PKC
, p42, p38
MAP kinases, and ARF in nuclei isolated from S-phase of the
regenerating liver cells were much higher than those of the control
nuclei (28 h after sham-operated liver). In contrast, the nuclear
PKC
II level of the regenerating liver cells was lower compared with
the control nuclei. Analysis of Western blotting of the AH7974 nuclei
revealed the presence of PKC isozymes (
,
I,
II, and
) (data
not shown).
Fig. 6.
Changes in PKCs and MAP kinases during liver
regeneration. A, the nuclei (50-200 µg of protein) from
28 h liver after partially hepatectomized (+) or sham-operated
() rat were separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted with various antibodies. A blot representative of three
independent experiments is shown. B, the isolated nuclei (50 µg of protein) during regenerating liver from partially
hepatectomized rats (2, 5, 12, 18, 20, 24, 26, 28, and 32 h
post-hepatectomy) were separated by SDS-polyacrylamide gel
electrophoresis and immunoblotted with various antibodies. Changes of
nuclear levels of PKCs and MAP kinases (MAPK) were
quantified by scanning densitometry and mass of each PKC and MAP kinase
isozyme was designated as 100%. Results are given as mean of three
different experiments.
[View Larger Version of this Image (22K GIF file)]
level
in the S-phase cell nuclei. On the other hand, the level of PKC
was not changed, and the PKC
II level rather decreased during liver regeneration. Significant increases in the nuclear levels of p42 and
p38 MAP kinases were observed in S-phase.
Fig. 7.
Western blot (A) and amount of G
proteins (B) in the nuclei from AH7974, rat resting liver,
and regenerating liver. The nuclei (300 µg of protein) isolated
from AH7974 (lane 1), rat resting liver (lane 2),
and rat regenerating liver (28 h after partial hepatectomy) (lane
3) were separated on a 13% SDS-polyacrylamide gel electrophoresis
and immunostained with the indicated antibodies. A blot representative
of three independent experiments is shown. B, amount of G
proteins of various nuclei shown in A was quantified by
scanning densitometry and mass of all nuclei (AH cells, rat resting
liver cells (Go), and rat regenerating liver cells
(S)) of each G protein was designated as 100%. Results are
shown as mean ± S.E. from three independent experiments.
[View Larger Version of this Image (44K GIF file)]
S-dependent and oleate-dependent, were
present in the isolated nuclei from rat resting, regenerating liver
cells, and rat ascites hepatoma AH7974 cells. Their
GTP
S-dependent PLD activities were markedly stimulated
by rARF. The nuclear ARF-dependent PLD activity was very
high in the AH7974 cells, and its elevation was observed at S-phase of
the regenerating liver cells. The nuclei isolated from the regenerating
liver and AH cells contained ARF, RhoA, Cdc42, and various PKC isozymes
(PKC
, PKC
II, and PKC
). The ARF-dependent PLD has
been known to be activated synergistically by either RhoA, Cdc42, or
PKC
(21, 22, 32, 34, 35). Balboa et al. (13, 23) have
reported that isolated nuclei from MDCK cells contain an
ATP-dependent PLD activity in the presence of GTP
S was
inhibited by protein kinase C inhibitors (chelerythrine and calphostin
C) and ADP-ribosylation of RhoA by C3 exoenzyme, indicating that the
MDCK nuclear PLD activity is synergistically regulated via RhoA and
protein kinase C. However, we have not observed synergy in the nuclear
ARF-dependent PLD activities of both rat liver cells and
AH7974 cells by either ATP or PMA when assayed with an exogenous
substrate (PE/PIP2/PC vesicles), although considerable
PKC
was present in these nuclei. C3 exoenzyme is known to block the
binding to effector enzymes by ADP-ribosylating Rho proteins (RhoA,
RhoB, RhoC) (36). Recently, Clostridium difficile toxin B
has been found to be available for analyzing the role of Rho family
proteins in PLD activation (37, 38). Toxin B is monoglucosyltransferase
catalyzing the incorporation of glucose into threonine residue at
position 37 of Rho family proteins (Rho, Rac, Cdc42) (39). The
ARF-dependent nuclear PLD activities of the hepatic cells
were partially inhibited by toxin B, but not by C3 exoenzyme,
suggesting that other Rho family protein rather than RhoA (most likely
Cdc42) may be involved in the nuclear ARF-dependent PLD
activity of rat hepatic cells (data not shown). Thus the rat liver
nuclear PLD may be different from the MDCK nuclear enzyme in lower
activation by PKC and insensitivity to C3 exoenzyme.
S-dependent PLD
activity in nuclei is stimulated by translocation of cytosolic ARF to
nuclei. This possibility could be supported by our finding that level of the nuclear ARF was increased in the S-phase and were higher in the
AH7974 nuclei. Second, certain PLD is newly expressed by receptor
activation by various stimulants produced after hepatectomy. The level
of PLC
4 in nuclei has been known to increase in liver regeneration
(6). Finally, translocation of PLD to nuclei occurs in response to
external stimuli induced by hepatectomy. Recent reports have
demonstrated that cPLA2 and MAP kinases also are translocated to nuclear envelope by cell stimulation with growth factors and A23187 (43-45). However, the mechanism underlying the transient elevation of the nuclear ARF-dependent PLD
activity in the rat-regenerating liver remains to be disclosed.
is
located in the nuclei from unstimulated liver (47). We have
demonstrated in this study that various levels of PKC isozymes, high
levels of PKC
and PKC
II and low level of PKC
, were present in
the isolated nuclei from rat resting liver cells and that the nuclear
PKC
level, but not PKC
II, was increased during S-phase. These
results lead us to assume that nuclear translocation of the PKC
during S-phase may result from increase of nuclear DG levels produced
by activation of the nuclear ARF-dependent PLD.
*
This work was supported in part by a grant-in-aid for
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§
To whom correspondence should be addressed: Dept. Biochemistry,
Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan.
Tel.: 81-58-267-2228; Fax: 81-58-265-9002.
1
The abbreviations used are: PI,
phosphatidylinositol; DG, diacylglycerol; PC, phosphatidlycholine;
DPPC, dipalmitoylphosphatidylcholine; PE, phosphatidylethanolamine;
PIP2, phosphatidylinositol 4,5-bisphosphate; PMA,
phorbol 12-myristate 13-acetate; PLD, phospholipase D; PLC, phospholipase C; PBut, phosphatidylbutanol; ARF, ADP-ribosylation factor; rARF, recombinant ADP-ribosylation factor; GTPS, guanosine 5
-O-(3-thiotriphosphate); AH, ascites hepatoma; MAP,
mitogen-activated protein; PKC, protein kinase C; MDCK, Madin-Darby
canin kidney.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.