Phospholipase C (PLC) (
)catalyzes a critical step in
the signaling systems of a variety of physiological stimuli through the
hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP
)
to inositol 1,4,5-trisphosphate and diacylglycerol, both of which act
in cells as second messengers. Inositol 1,4,5-trisphosphate mobilizes
Ca
from intracellular stores, and diacylglycerol
activates protein kinase C, leading to various cellular responses
(Berridge and Irvine, 1984; Majerus, 1992; Nishizuka, 1984). These
events seem to regulate not only short term cellular responses but also
longer term responses such as cell growth and differentiation (Berridge
and Irvine, 1989). So far, nine PLC cDNAs have been cloned from
mammalian cells and can be divided into three types,
,
, and
, on the basis of their structures (Rhee et al., 1989;
Kritz et al., 1991; Lee et al., 1993; Jhon et
al. 1993). The existence of multiple forms of PLC suggests that
each isozyme may differ in tissue distribution, intracellular location,
regulatory mechanism, and further downstream function. In fact, it has
been shown that each PLC distributes differently and couples to
different signaling systems. The
-type isozymes are activated by
GTP binding proteins such as Gq
and the 
subunits of G
proteins (Taylon et al., 1991; Smrcka et al., 1991;
Camp et al., 1992; Katz et al., 1992). On the other
hand,
-type isozymes have been shown to be activated through
receptor activation encoding tyrosine kinases (Wahl et al.,
1989; Meisenhelder et al., 1989; Margolis et al.,
1989; Morrison et al., 1990; Kim et al., 1991).
However, the regulatory mechanisms of PLC
isozymes remain
unclear.
Recently, polyphosphoinositide turnover has been
demonstrated to occur in the nucleus as well as in the plasma membrane
during cell growth and differentiation (Cocco et al., 1987,
1989; York and Majerus, 1994; Divecha et al., 1989). Moreover,
much evidence has shown that a PIP
-hydrolyzing activity
exists in the internal nuclear matrix and that PIP
synthesis activity is present in the envelope-depleted nucleus (Divecha et al., 1991; Payrastre et al., 1992; Capitani et
al., 1990). These results suggest the involvement of PLC in
nuclear function, probably playing a key role in gene expression and
DNA synthesis. The signaling mechanisms of the receptor-mediated
activation of PLC, which takes place around plasma membranes, are well
established, at least for
-type and
-type isozymes.
Concerning PLCs in the nucleus, PLC
1 has been shown to be present
in the nuclei of Swiss 3T3 cells, in which polyphosphoinositides are
hydrolyzed quickly in response to insulin growth factor-1 (IGF-1),
although there is no direct evidence that the breakdown is induced by
nuclear PLC
1 (Martelli et al., 1992). More recently,
Asano et al.(1994) have presented evidence that there is a new
PLC in the nucleus. They purified a nuclear PLC from rat ascites
hepatoma cells (AH 7974). The PLC isozyme was only detected in the
nuclei of regenerating liver, not in adult resting liver cells.
Independently, we have been trying to isolate a new PLC cDNA clone
expressed dominantly in growing cells and present in the nucleus using
a cDNA library prepared from regenerating liver.
Here, we report the
molecular cloning of a novel PLC, PLC
4, which localizes in
nuclei, and present evidence that the expression of PLC
4 is very
high in growing cells and induced in S-phase during the cell cycle.
EXPERIMENTAL PROCEDURES
Materials and
Cells
Phosphatidyl[2-
H]inositol
4,5-bisphosphate (173.9 Mbq/µmol) was purchased from DuPont NEN.
Phosphatidylinositol 4,5-bisphosphate was prepared by the method of
Schacht(1978). [
-
P]Deoxycytidine
triphosphate (dCTP) was from Amersham Corp. Anti-PLC
1 and
1
antibodies were produced by the methods described before (Homma et
al., 1990). Anti-PLC
1 antibody was from UBI. Reverse
transcriptases were from Life Technologies, Inc. Rat 3Y1, Src/3Y1, RLH
84, RH 34, and Swiss 3T3 cells were from the Japanese Cancer Research
Resources Bank. They were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum.
Isolation of mRNA from Regenerating Rat
Liver
Regenerating livers were prepared from partially
hepatectomized rats (Donryu strain) 24 h after the removal of
approximately two-thirds of liver mass according to the method of
Higgins and Anderson(1931). The poly(A)
RNA was
isolated by the guanidine method (Han et al., 1987) and
Oligotex-(dT)30 (Roche, Japan).
Reverse Transcriptase PCR
A total of 6
µg of RNA from regenerating livers was used in 120 µl of the
oligo(dT)-primed reverse transcription reaction mixture. The cDNA
products were used as a source of polymerase chain reaction templets.
Three sensed primers responding to the highly conserved X domain
sequences (R/K)I(I/L/V)(I/V)K(N/H/G)KK of
-,
-, and
-type PLCs, respectively, and one antisense primer responding to
the Y domain DSSNY(M/D/S/N)P were synthesized. The nucleotide sequences
of the primers were as follows:
5`-GACAAGCTTAT(C/T)(C/T)TI(A/G)TIAA(A/G)AA(C/T)AA(A/G)AA-3` (P-Xb),
5`-GACAAGCTTAAGAT(C/T)(A/C)T(C/G/T)AT(C/T)AA(A/G)CAIAA(A/G)AA3` (P-Xg),
5`-GACAAGCTTAAG(A/G)TCCTI(C/G/T)T(C/G)AA(A/G)GGIAA(A/G)AA-3` (P-Xd),
and 5`-ATGGGATCCIGG(C/G)(A/T)(C/T)(A/G)TA(A/G)TT(C/T)GAIGA(A/G)TC-3`
(P-Y). The underlined sequences at the 5`-end of each oligonucleotide
indicate restriction enzyme cleavage sites (HindIII and BamHI sites for the sense and antisense primers,
respectively); three nucleotides (GAC or ATG) were added to facilitate
cutting by the enzymes. To amplify the DNA, three 40-cycle PCRs were
performed. A 420-bp PCR product was further subcloned into pBluescript
sequencing vector. The nucleotide sequences were determined by the
autocycle sequencing method using an A.L.F. DNA sequencer (Pharmacia
Biotech Inc.). The 420-bp clone coding a new PLC DNA fragment was
identified.
Construction of a Regenerating Rat Liver cDNA
Library
Oligo(dT)-primed cDNA from regenerating rat liver
was synthesized using a
-ZAP cDNA synthesis kit (Stratagene), and
the different sizes were fractionated on 1.0% agarose gel. Purification
was done with Geneclean II (Bio 101, Inc.). The cDNA >1.2 kb was
cloned into Uni-ZAP vector. The original library contained over 3.6
10
independent recombinant clones.
Library
Screening
[
P]dCTP-labeled probes
were prepared from a 420-bp PCR product by a random priming procedure
and used to screen the regenerating rat liver cDNA library. Among 8
10
clones screened, a positive bacteriophage
containing a 2.7-kb insert was isolated. The insert was cloned into
pBluescript, and sequences were determined by the dideoxy
chain-termination method with [
P]dCTP labeling.
Northern Hybridization and
RT-PCR
Poly(A
) RNA (5 µg) was
subjected to 1% formaldehyde gel and transferred onto a nylon membrane
(GeneScreen Plus, DuPont NEN). A 2-kb EcoRI-EcoRI
fragment was labeled with [
-
P]dCTP and used
as a probe. The membranes were hybridized for 20 h in 50% formamide
solution containing 5
SSPE, 5
Denhardt's, 0.1%
SDS, and 100 µg/ml herring sperm DNA using the labeled probes. To determine changes in PLC
4 mRNA levels, 0.1 µg of mRNA
prepared from cells at the indicated times was used for RT-PCR using
the primers 5`-GACAAGCTTGTGCTGAGCAAGGATGGC-3` and
5`-AAAGGATCCTGGTCTTCTGCTGTTCCC-3` in the presence of
[
-
P]dCTP. Considering the linearity of the
reaction, 30 cycles was chosen. The resulting products were subjected
to electrophoresis in 5% polyacrylamide gels and autoradiography.
Expression of the PLC
4 Whole Coding Region in
Escherichia coli
PLC
4 was constructed into expression
vector pGEX-3X. Because a stop codon exists just before the
translational initiation site, PCR was used to synthesize the DNA
fragment from the beginning of the coding region to the first BamHI site (at 335 bp). To facilitate the construction, a SpeI site was added to the sense primer. The PLC
4 cDNA
was then constructed into the HindIII linker containing
pGEX-3X by flanking the whole coding region (SpeI-XhoI) with the HindIII linker.
Glutathione S-transferase (GST)-PLC
4 fusion protein was
expressed in JM109 E. coli. After a 4-h preculture at 37
°C and a 5-h induction with
isopropyl-1-thio-
-D-galactopyranoside at 25 °C, the
bacteria were lysed in sodium cholate lysis buffer (2% sodium cholate,
50 mM Tris/HCl (pH 7.6), 1 mM EDTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl
fluoride (PMSF), and 0.1 mM diisopropyl fluorophosphate). The
lysates were sonicated and ultracentrifuged at 100,000
g for 1 h. The soluble GST-PLC
4 fusion proteins were then
purified on a glutathione-Sepharose column.
PLC Activity Assay
PLC activity was
assayed using [
H]PIP
as a substrate
by the method described previously (Kato et al., 1992).
Phospholipid vesicles were prepared by solubilizing 40 mM phosphatidylethanolamine and 0.1 mM PIP
(10
dpm) in chloroform/methanol (2:1 (v/v)). The mixture
was dried under a nitrogen stream, suspended in 1 ml of 0.2 M KCl, and then sonicated for about 30 s. PLC assays were carried
out in a solution containing 50 mM Mes/NaOH (pH 6.5), 10
µM CaCl
, 1 mg/ml bovine serum albumin, 20,000
dpm [
H]PIP
(200 µmol), and
purified GST-PLC
4 fusion protein. The reaction solution was
incubated in 37 °C for 2 min, and the reaction was stopped by
adding 2 ml of chloroform/methanol (2:1 (v/v)). Inositol trisphosphates
were extracted with 0.5 ml of 1 N HCl, and the radioactivity
in the upper aqueous phase was measured.
Production and Purification of Polyclonal
Anti-PLC
4 Antibody
PLC
4 cDNA fragment (EcoRI-XhoI) was constructed into pGEX-3X expression
vector. The overexpressed GST-PLC
4 fusion proteins were used as
the source of antigen. The fused proteins were lysed with a lysis
buffer composed of 40 mM Tris/HCl (pH 7.6), 5 mM EDTA, 0.1 mM PMSF, 0.1 mM diisopropyl
fluorophosphate, 1% Triton, 0.5% deoxycholate, and 0.1% SDS. The
lysates were applied to a glutathione-Sepharose column and eluted with
50 mM Tris/HCl (pH 8.0) and 50 mM glutathione. To
obtain the pure antigen, disk electrophoresis (Nihon Eido NA-1800) was
further carried out. The fractions at 44 kDa were collected and
lyophilized. The antigen (200 µg) was mixed with complete
Freund's adjuvant (Difco) for the first and second immunizations
and thereafter with incomplete Freund's adjuvant. The rabbit
received booster immunizations every 2 weeks. After immunizing five
times, the serum was collected. The antibody was purified in three
steps. First, the materials precipitated by 50% saturated ammonium
sulfate were collected. Second, after the precipitates were dissolved
in 20 mM Tris/HCl (pH 7.4) and applied to GST-coupled
glutathione-Sepharose, the void fraction was collected. Third, the
eluates were further purified as follows. Full-length PLC
4
overexpressed in E. coli was used as the material for
immunoaffinity purification. The purified GST-PLC
4 was
electrophoresed in a 7% SDS-polyacrylamide gel electrophoresis gel and
then transferred to a nitrocellulose membrane (ATTO, Japan) by Western
blotting. GST-PLC
4 bands were cut into small pieces and mixed
with the antibody. The mixture was agitated by gentle shaking at 4
°C for 30 min. The bands were washed three times with PBS, and the
anti-PLC
4-specific antibody was eluted from the membranes with
0.1 M glycine/HCl (pH 2.7) and neutralized immediately with
1.0 M Tris/HCl (pH 9.0).
Preparation of Cell Fractions and Western
Blotting
Nuclear and cytoplasmic fractions of Swiss 3T3
cells were prepared as described by Divecha et al.(1991).
Swiss 3T3 cells were harvested with 0.2% trypsin and washed two times
with cold PBS. The cells were suspended in buffer A (10 mM Tris/HCl (pH 7.8), 1% Nonidet P-40, 10 mM
-mercaptoethanol, 0.5 mM PMSF, 1 µg/ml aprotinin
and leupeptin) for 2 min on ice. An equal volume of distilled
H
O was added, and the cells were allowed to swell for 2
min. The cells were sheared by ten passages through a 22-gauge needle.
The nuclei were recovered by centrifugation at 400
g for 6 min and washed once with buffer B (10 mM Tris/HCl
(pH 7.4), 2 mM MgCl
, 0.5 mM PMSF, 1
µg/ml aprotinin and leupeptin). The cell lysates from different
time points were adjusted to an equal cell number/ml. Then, equal
volumes of lysates were subjected to 7.5% SDS-polyacrylamide gel
electrophoresis, and proteins were transferred to nitrocellulose
membranes. The membranes were incubated with 1:500 diluted anti-PLC
4 antibody and stained with alkaline phosphatase-conjugated
anti-rabbit IgG.
Immunofluorescent Staining
Swiss 3T3
cells were cultured on glass coverslips. The cells were serum-starved
for 30 h in Dulbecco's modified Eagle's medium containing 1
mg/ml bovine serum albumin, 5 µg/ml insulin, 5 µg/ml
transferrin, and sodium selenite (5 ng/ml) and then stimulated by
adding 10% fetal calf serum for the indicated times. The cells were
washed with PBS, fixed with 3.7% formaldehyde in PBS for 20 min,
permeabilized with 0.2% Triton X-100 in PBS for 5 min, and washed with
PBS. The cells were incubated with 1:25 diluted anti-PLC
4
antibody for 90 min and washed three times with PBS. Then, they were
incubated for 40 min with 1:40 diluted second antibody conjugated to
fluorescein isothiocyanate and washed three times. The stained cells
were observed with Axioskop (Zeiss, Germany).
RESULTS
Isolation of PLC
4 cDNA Clones
It
is very important to select a good gene pool containing many high
copies of the gene to be searched. Regenerating rat liver was chosen as
the gene source for this research because it was thought to provide a
good model for elucidating the relationship between cell proliferation
and polyphosphoinositide turnover in the nucleus. We isolated mRNA from
regenerating rat liver 24 h after partial hepatectomy and carried out
RT-PCR. The primers were designed according to the highly conserved
amino acid sequence in the X and Y domains with consideration given to
the codon usage and notable amino acid similarity among PLC isozymes.
We obtained several products. Among them, a 420-bp PCR product
contained a PLC-like sequence distinct from those of known PLC
isozymes. Therefore, we screened the regenerating rat liver cDNA
library with the 420-bp PCR product and obtained one positive clone,
named 7A. Clone 7A had a 2.7-kb insert. Complete sequencing of clone 7A
revealed an open reading frame of 2310 bp surrounded by 135 bp of
5`-noncoding sequence and 102 bp of 3`-noncoding sequence (Fig. 1). The initiation methionine was designated at position
136 because there was an in-frame stop codon upstream of this
methionine; comparing the sequence with known PLCs further supports
this designation. The 3`-noncoding region contained a poly(A) tail, but
no polyadenylation addition signal was found. The deduced amino acid
sequence of clone 7A revealed an overall structure similar to the PLC
type. Thus, the protein encoded by the 7A clone was named PLC
4. PLC
4 consists of 770 amino acids with a calculated
molecular size of 85 kDa. The amino acid sequence of PLC
4 is more
similar to that of PLC
2 than to PLC
1 or PLC
3,
especially in the region extending from the amino-terminal to the
beginning of the X domain (Fig. 2). Both PLC
4 and PLC
2 have possible nuclear translocation signal peptide sequences
near the amino-terminal. This probably indicates a functional
similarity between PLC
4 and PLC
2. A PH domain and an
EF-hand-like domain are also included in PLC
4.
Figure 1:
Nucleotide sequence of PLC
4 and
the deduced amino acid sequence. Nucleotide and amino acid sequence
numbers are shown on the left and right,
respectively. The DNA sequence is numbered starting with the
translation initiation codon. The translation termination codon is
marked by an asterisk. The EF-hand-like domain is double
underlined. The possible nuclear translocation signal (amino acids
25-36) is written in shadowed letters, and the dotted line indicates the PH domain peptide
sequence.
Figure 2:
Linear representation of PLC
4, PLC
1, PLC
2, and PLC
3. Open boxes X and Y denote the conserved sequences found in all mammalian PLCs. The numbers above each box refer to the first and last
amino acid. The extent of sequence identity to the corresponding region
of PLC
4 is indicated by the
percentages.
To confirm that
clone 7A encodes an inositol phospholipid-specific PLC, the whole
coding region of clone 7A was subcloned into pGEX-3X expression vector
and overexpressed in E. coli. The soluble GST-PLC
4
fusion proteins were purified on a glutathione-Sepharose column.
GST-PLC
4 showed PIP
-hydrolyzing activity. The
specific activity of GST-PLC
4 was found to be 1.6 µnol/min/mg
protein, indicating that it was a PLC isozyme.
Distribution of PLC
4 and Its Expression in
Growing Cells
Northern hybridization was carried out to
detect the distribution of PLC
4 in different tissues and to
compare its expression in normal liver and regenerating liver. The
results are shown in Fig. 3a. PLC
4 was expressed
abundantly in regenerating liver, while it was only slightly detected
in normal liver. The expression was also very high in intestine and
moderately high in thymus. Since the cells in both intestine and
regenerating liver have high proliferating activities, it is reasonable
to suppose that PLC
4 is related to cell growth and cell
proliferation. To elucidate whether this is a general phenomenon or
just specific to regenerating liver and intestine, we investigated the
expression of PLC
4 in four cell lines, RL-34, RhL-84, 3Y1, and
Src/3Y1 (Fig. 3b). RL-34 is a rat hepatocyte cell line,
RhL-84 is a rat hepatoma cell line, and Src/3Y1 is src-transformed 3Y1 cells. When comparisons were made between
RL-34 and RhL-84 and between 3Y1 and Src/3Y1, we found that PLC
4
is expressed more vigorously in hepatoma (RhL-84) and src-transformed cells (Src/3Y1). These data clearly show that
PLC
4 is closely related to cell proliferation.
Figure 3:
PLC
4 is highly expressed in
proliferating tissues and cells. a, distribution of PLC
4. RNA was extracted from various rat tissues, and Northern
analysis was carried out. b, High PLC
4 expression in
transformed cells. The expression levels of PLC
4 in different
cell lines were detected by Northern hybridization. src/3Y1 refers to src-transformed 3Y1 cells. RL-34 is a rat hepatocyte cell
line, and dRLh-84 is a rat hepatoma cell line. c,
serum-induced PLC
4 mRNA expression in Swiss 3T3 cells.
Serum-starved Swiss 3T3 cells were stimulated by adding 10% fetal calf
serum. At the indicated times, mRNA was isolated, and RT-PCR was
performed.
Serum Induction of PLC
4
Swiss 3T3
cells were starved in serum-free medium for 30 h and then stimulated by
adding 10% fetal calf serum. After serum stimulation for the indicated
times, changes in PLC
4 mRNA levels were determined by RT-PCR. Fig. 3c shows the effect of serum on the expression of
PLC
4. PLC
4 was expressed at low levels when the cells were
starved. The expression of PLC
4 mRNA began to increase 4 h after
serum stimulation and reached a maximum at 8 h. After that, PLC
4
mRNA levels decreased gradually until 20 h. We also examined the time
course of the changes in PLC
4 content during the cell cycle by
Western blotting. We prepared both nuclear and cytosol fractions from
Swiss 3T3 cells according to the methods described by Divecha et
al. (1991). The anti-PLC
4 antibody used for Western blotting
was affinity purified, and only a single band appeared at the expected
molecular weight when it was used to blot cell lysates. To check
whether the nuclear fraction was contaminated by cytosol proteins, we
examined for actin contamination as a control. The results are shown in Fig. 4. PLC
4 appeared mostly in the nuclear fraction,
while PLC
1 and
1 were in the cytosol. PLC
1 was
detected in the nucleus. Because actin was not detected in the nuclear
fraction, contamination from the cytosol fraction could be neglected.
Among the PLCs examined (PLC
1,
1,
1, and
4), only
the content of PLC
4 changed depending on the phase of the cell
cycle. PLC
4 was only slightly detected at G
-phase and
early G
-phase (0 and 6 h). Then, the level was elevated at
12 h and climbed further reaching a maximum at 16 h. The high level
continued during S-/M-phase (until 24 h). When the cells entered the
next G
-phase (28 h), the PLC
4 content decreased
dramatically. On the other hand, the level of other PLCs remained
stable during the cell cycle. These results imply that PLC
4 is
probably one of the early genes expressed during the transition from
G
- to S-phase and functions curing the S- and M-phases.
Figure 4:
Changes in PLC
4 content during the
cell cycle. Swiss 3T3 cells were cultured in serum-free medium for 30 h
and then stimulated with serum for various periods. The cell lysates
were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. The membranes were
immunoblotted with anti-PLC
4 antibody (a), anti-PLC
1 antibody (b), anti-PLC
1 antibody (c),
anti-PLC
1 antibody (d), or anti-actin antibody (e). Column N refers to the nuclear fraction, and
column C refers to the cytosol
fraction.
Localization of PLC
4 in Swiss 3T3
Cells
To confirm the localization of PLC
4 in Swiss
3T3 cells, cells in various stages of the cell cycle were stained for
immunofluorescent microscopy using an anti-PLC
4 antibody (Fig. 5). In resting cells (0 h), immunofluorescent density was
very slight. However, at 8 h after serum stimulation, the nuclear area
began to be stained, and the density increased up to 16 h while the
cytosolic area was not stained. This strong staining in the nucleus
continued to the end of M-phase. PLC
4 in the nuclear areas almost
disappeared at 28 h and began to increase again when cells re-entered
the next S-phase (36 h, data not shown). All these data show that PLC
4 is expressed during S-/M-phase and is located in nuclei.
Figure 5:
Immunofluorescent staining of
serum-stimulated Swiss 3T3 cells with anti-PLC
4 antibody.
Serum-starved Swiss 3T3 cells were stimulated by adding 10% fetal calf
serum. The cells were fixed at 0 h (A and a), 8 h( B and b), 16 h (C and c), 24 h (D and d), and 28 h (E and e). A-E are the immunofluorescent micrographs of cells with
anti-PLC
4 antibody; a-e are phase-contrast
micrographs.
DISCUSSION
We isolated PLC
4 from regenerating rat liver. This PLC
4 not only shows nuclear localization but also a strong
correlation with cell proliferation. It is highly expressed in
regenerating liver but was detected at low levels in normal liver. When
we investigated the distribution of PLC
4, we found that it is not
restricted to the regenerating liver but is also expressed strongly in
intestine, hepatoma, and src-transformed 3Y1 cells. These data
indicate that the distribution of PLC
4 is quite different from
that of other known PLCs. It appears to be expressed selectively in
cells with high proliferating activity.
A variety of data have shown
that polyphosphoinositide turnover also occurs in the nucleus and
changes dynamically during cell proliferation and differentiation
(Cocco et al., 1987, 1989; York Majerus, 1994; Divecha et
al., 1989, 1991). However, no direct evidence has been provided,
although many researchers have tried to find a PLC isozyme responsible
for polyphosphoinositide turnover in the nucleus. Recently, Martelli et al.(1992) reported that PLC
1 is present in the nuclei
of Swiss 3T3 cells and may be responsible for the breakdown of
polyphosphoinositide in the nucleus in response to IGF-1. In this case,
PLC
1 is activated immediately after IGF-1 stimulation, and the
activity returns to normal within 30 min. Therefore, it seems unlikely
that PLC
1 plays an important role in events during S-phase and
later stages of the cell cycle. PLC
4 behaves differently from PLC
1 since its expression is induced by mitogens and its level
reaches a maximum at S-phase. During the cell cycle, the levels of PLC
1 and PLC
1, which are mostly located in the cytosol, did not
change. Similarly, PLC
1 levels did not change significantly,
although it is present in the nucleus.
PLC
4 is highly
homologous to PLC
2 with 69% identity in the amino acid sequence
(Meldrum et al., 1989, 1991). However, the functions and
distribution of PLC
2 have not been investigated yet. PLC
2
may play a similar role to PLC
4.
The increased hydrolysis of
nuclear polyphosphoinositides during S-phase of the cell cycle has been
observed in regenerating liver (Kuriki et al. 1992) and HeLa
cells (York and Majerus, 1994), while levels of cytoplasmic
polyphosphoinositides remain constant in HeLa cells. There are at least
three possible explanations for the increase in nuclear PLC activity.
First, PLC in nuclei may be activated by some signaling molecules. This
may be the case for PLC
1 activation upon treatment with IGF-1.
Second, translocation of PLC from the cytosol to nucleus may occur in
response to stimuli. Finally, a certain PLC isozyme may be newly
synthesized in response to stimuli. Our results on time course changes
of PLC
4 suggest that the last event probably occurs in cells
because the expression of PLC
4 increases dramatically at S-phase.
It has been reported that the treatment of nuclear matrix with PLC
results in a release of nucleic acid, implying that inositol
phospholipids are responsible for the hydrophobic interaction between
nuclear matrix and nucleic acids (Cocco et al., 1980).
Therefore, the dramatic increase in PLC
4 at S-phase may influence
the interaction between nuclear matrix and nucleic acids. It is also
likely that PLC
4 may activate a certain protein kinase C through
diacylglycerol formation in the nucleus, causing the phosphorylation of
lamin and the activation of DNA polymerase and topoisomerase leading to
cell proliferation (Divecha et al., 1989). Since PDGF
stimulation of NIH 3T3 cells (Fields et al., 1990) and IGF-1
stimulation of Swiss 3T3 cells (Divecha et al., 1991) induce
the translocation of protein kinase C from the cytosol to nucleus,
translocated protein kinase C rather than pre-existing protein kinase C
may play a crucial role in the nucleus. Furthermore, Sylvia et
al.(1988) provided direct evidence that a hydrolyzed product of
polyphosphoinositides, inositol 1,4-bisphosphate, in the nucleus could
directly regulate DNA synthesis through an increase in DNA polymerase
activity. PLC
4 may activate DNA polymerase
by
producing inositol 1,4-bisphosphate during S-phase of the cell cycle.
It will be very interesting to identify the relationship between the
increase in PLC
4 activities and DNA polymerase activation in
S-phase, but it remains to be solved in the future.