A new phospholipase C delta 4 is induced at S-phase of the cell cycle and appears in the nucleus.

To discover a new phospholipase C (PLC) related to cell growth, we screened a cDNA library prepared from regenerating rat liver. A novel PLC (PLC delta 4) encoding a polypeptide of 770 amino acids with structural similarity to PLC delta-type isozymes was isolated. PLC delta 4 mRNA is expressed more remarkably in regenerating liver than in normal resting liver. It is also distributed abundantly in tumor cells such as hepatoma and src-transformed cells. Furthermore, its expression can be induced markedly by serum treatment and reaches a maximum at 8 h. Western blot analysis and immunocytochemical staining showed that PLC delta 4 is dominantly present in nucleus. Nuclear PLC delta 4 dramatically increases at the transition from G1- to S-phase, and the high content continues to the end of M-phase. PLC delta 4 almost disappears when cells re-enter the next G1-phase. On the other hand, the contents of PLC beta 1, PLC gamma 1, and PLC delta 1 do not change significantly during the cell cycle. These results suggest that PLC delta 4 is expressed in nucleus in response to mitogenic stimulation and plays important roles in cell growth as one of the early genes expressed during the transition from G1- to S-phase in the cell cycle.

To discover a new phospholipase C (PLC) related to cell growth, we screened a cDNA library prepared from regenerating rat liver. A novel PLC (PLC ␦4) encoding a polypeptide of 770 amino acids with structural similarity to PLC ␦-type isozymes was isolated. PLC ␦4 mRNA is expressed more remarkably in regenerating liver than in normal resting liver. It is also distributed abundantly in tumor cells such as hepatoma and src-transformed cells. Furthermore, its expression can be induced markedly by serum treatment and reaches a maximum at 8 h. Western blot analysis and immunocytochemical staining showed that PLC ␦4 is dominantly present in nucleus. Nuclear PLC ␦4 dramatically increases at the transition from G 1 -to S-phase, and the high content continues to the end of M-phase. PLC ␦4 almost disappears when cells re-enter the next G 1 -phase. On the other hand, the contents of PLC ␤1, PLC ␥1, and PLC ␦1 do not change significantly during the cell cycle. These results suggest that PLC ␦4 is expressed in nucleus in response to mitogenic stimulation and plays important roles in cell growth as one of the early genes expressed during the transition from G 1 -to S-phase in the cell cycle.
Phospholipase C (PLC) 1 catalyzes a critical step in the signaling systems of a variety of physiological stimuli through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to inositol 1,4,5-trisphosphate and diacylglycerol, both of which act in cells as second messengers. Inositol 1,4,5-trisphosphate mobilizes Ca 2ϩ 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 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(Cocco et al., , 1989York and Majerus, 1994;Divecha et al., 1989). Moreover, much evidence has shown that a PIP 2 -hydrolyzing activity exists in the internal nuclear matrix and that PIP 2 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.
[␣-32 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/ * This work was supported in part by grants for Science Research and for Cancer Research from the Ministry of Education, Science, and Culture of Japan and for Aging and Health from the Ministry of Health and Welfare, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D50455.
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) ϩ RNA was isolated by the guanidine method (Han et al., 1987) and Oligotex-(dT)30 (Roche, Japan).
Reverse Transcriptase PCR-A total of 6 g of RNA from regenerating livers was used in 120 l 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: 5Ј-GACAAGCT- The underlined sequences at the 5Ј-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 Ͼ1.2 kb was cloned into Uni-ZAP vector. The original library contained over 3.6 ϫ 10 6 independent recombinant clones.
Library Screening-[ 32 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 ϫ 10 5 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 [ 32 P]dCTP labeling.
To determine changes in PLC ␦4 mRNA levels, 0.1 g of mRNA prepared from cells at the indicated times was used for RT-PCR using the primers 5Ј-GACAAGCTTGTGCTGAGCAAGGATGGC-3Ј and 5Ј-AAAGGATCCTGGTCTTCTGCTGTTCCC-3Ј in the presence of [␣-32 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 g/ml aprotinin, 10 g/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM diisopropyl fluorophosphate). The lysates were sonicated and ultracentrifuged at 100,000 ϫ 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 [ 3 H]PIP 2 as a substrate by the method described previously (Kato et al., 1992). Phos-pholipid vesicles were prepared by solubilizing 40 mM phosphatidylethanolamine and 0.1 mM PIP 2 (10 6 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 M CaCl 2 , 1 mg/ml bovine serum albumin, 20,000 dpm [ 3 H]PIP 2 (200 mol), 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 g) 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 g/ml aprotinin and leupeptin) for 2 min on ice. An equal volume of distilled H 2 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 ϫ g for 6 min and washed once with buffer B (10 mM Tris/HCl (pH 7.4), 2 mM MgCl 2 , 0.5 mM PMSF, 1 g/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 g/ml insulin, 5 g/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).

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 5Ј-noncoding sequence and 102 bp of 3Ј-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 3Ј-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.
To confirm that clone 7A encodes an inositol phospholipidspecific 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 2 -hydrolyzing activity. The specific activity of GST-PLC ␦4 was found to be 1.6 nol/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 srctransformed cells (Src/3Y1). These data clearly show that PLC ␦4 is closely related to cell proliferation.
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 0 -phase and early G 1 -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 1 -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 1 -to S-phase and functions curing the Sand M-phases.
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. 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 FIG. 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.
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(Cocco et al., , 1989York Majerus, 1994;Divecha et al., 1989Divecha et al., , 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(Meldrum et al., , 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 Sphase 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 in- FIG. 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. crease 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.