Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus*

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKγ came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKγ is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKγ didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKγ acts as a nuclear transport signal. Furthermore, to address the function of DGKγ in the nucleus, we produced stable cell lines of wild-type DGKγ and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKγ were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKγ regulates cell cycle.


Diacylglycerol (DG)
is a second messenger regulating various cellular responses (1,2). One of the important roles of DG is an activating of protein kinase C (PKC) (1,3,4). DG is physiologically produced as a result of the signal-induced hydrolysis of phosphatidylinositol by phospholipase C. The generated DG is phosphorylated to phosphatidic acid by diacylglycerol kinase (DGK) or metabolized by DG lipase (2,5,6). Thus, DGK is an important enzyme to inactivate PKC by reducing the DG level, contributing to regulating of the cellular response. In addition, phosphatidic acid itself activates PKC (7), phosphatidylinositol 4-phosphate 5-kinase (8,9), and mammalian target of rapamycin (10), and modulates Ras GTPase-activating protein (11).
Molecular cloning studies revealed that mammalian DGK family consists of at least nine subtypes (2). Although all DGKs have cysteinerich repeats similar to the C1A and C1B domains of PKCs in the N terminus and a catalytic domain in the C terminus, they are divided into five groups on the primary structure of these DGKs. Type I DGKs, including DGK␣, -␤, and -␥, have EF-hand motifs and two cysteine-rich regions (C1 domain) in the regulatory domain (12,13), whereas Type II DGKs, DGK␦ and -, have a pleckstrin homology domain instead of the EF-hand motif in addition to the C1 domain (14,15). The separated catalytic domains of DGK␦ and -are characteristic of Type II DGK. Type III, DGK⑀, has only one C1 domain in the regulatory domain (16). Myristoylated alanine-rich protein kinase C substrate phosphorylation site-like region and four ankyrin repeats are unique domains in Type IV DGK (17,18). The final group, type V, includes DGK, which has three cysteine-rich regions and a pleckstrin homology domain with overlapping Ras-associating domain (19). The DGKs are thought to be involved in development, differentiation, construction of neural network and immunity, etc. However, subtype specific function and regulation mechanisms of DGKs are not clear. Several groups have reported many different localization and translocation of DGKs, possibly contributing to their subtype-specific functions. In an effort to elucidate the function of DGK, we unexpectedly found that GFP-fused DGK␥ (GFP-DGK␥) became localized in the nucleus as well as the cytoplasm a few days after transfection but was localized mainly in the cytoplasm just after expressed in CHO-K1 cells. Although nuclear transportation of DGK␥ has never been reported, expression of DGK and DGK in the nucleus has been already described (20,21). In addition, DGK is thought to be involved in the regulation of cell cycle (21). These findings, together with the facts that phosphatidylinositol turnover exists within the nucleus and DG may be involved in the regulation of cell cycle (22)(23)(24)(25)(26)(27), suggest that DGK␥ has some physiological function in the nucleus. However, mechanism of the nuclear transportation and physiological functions of DGK␥ are unknown. We, therefore, investigated molecular mechanism and physiological significance of nuclear transportation of DGK␥.

EXPERIMENTAL PROCEDURES
Materials-CHO-K1 cells were donated from Dr. M. Nishijima (National Institute of Health, Tokyo, Japan). COS-7 cells, SH-SY5Y cells, and HeLa cells were purchased from the RIKEN Cell Bank. Fetal bovine serum, RNase A, and anti-FLAG M2 monoclonal antibody were obtained from Sigma. FuGENE 6 Transfection reagent was obtained from Roche Applied Science. Propidium iodide was obtained from Wako (Osaka, Japan). FluoroLink Cy3-labeled goat anti-mouse IgG was purchased from Amersham Biosciences. We produced anti-GFP anti-*This work was supported by grants from the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from a grant-in-aid for Scientific Research on Priority Areas "Molecular Brain Science" and "Nuclear Dynamics" from the Ministry of Education, Culture, Sports, Science, and Technology in 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. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 To whom correspondence may be addressed. Cell Culture-CHO-K1 cells were cultured in Ham's F-12 medium (Nacalaitesque, Japan). COS-7 and NIH3T3 cells were cultured in Dulbecco's modified Eagles' medium (Nacalaitesque, Japan). SH-SY5Y cells were cultured in Dulbecco's modified Eagles' medium/Ham's F-12 medium (1:1) (Invitrogen). All cells were cultured at 37°C in humidified atmosphere containing 5% CO 2 . All media contained 25 mM glucose, and all were buffered with 44 mM NaHCO 3 and supplemented with 10% fetal bovine serum (Sigma), penicillin (100 units/ml), and streptomycin (100 g/ml) (Invitrogen). The fetal bovine serum used was not heatinactivated. The media for SH-SY5Y cells was added with 1% GlutaMA-X TM -I Supplement (Invitrogen).
Constructs of Plasmids Encoding DGK␥-fused GFP and Mutants-The constructs encoding DGK␥ having GFP at its N terminus (GFP-DGK␥) or at C terminus (DGK␥-GFP) and mutants having substitution of Cys-285 to Gly in the C1A domain (GFP-DGK␥ C1Am) or Cys-348 to Gly in the C1B domain (GFP-DGK␥ C1Bm) were previously described (28). The constructs encoding the mutants lacking the C1A or C1B domain (GFP-DGK␥ ⌬C1A or GFP-DGK␥ ⌬C1B) were also described previously (29). Kinase negative mutant of DGK␥ (GFP-DGK␥ KN) was produced by substitution of Gly-491 to Asp. Briefly, using the plasmid encoding GFP-DGK␥, site-directed mutagenesis was performed according to the manufacturer's recommended protocol with an ExSite PCR-based site-directed mutagenesis kit (Stratagene). The primers were 5Ј-GCTGGATCCTGGATTGCTTTGACAAGG-3Ј and 5Ј-CA-ACTGTGTCATCTCCGCCACAGGCAA-3Ј. The mutation was confirmed by verifying its sequence. Furthermore, to make kinase-negative mutants having substitution of Cys-285 or Cys-348 to Gly (GFP-DGK␥ KNC1Am and GGFP-DGK␥ KNC1Bm), the cDNA of DGK␥ KN was digested with SalI and SmaI and then subcloned into the SalI and SmaI site in the plasmid encoding GFP-DGK␥ C1Am and GFP-DGK␥ C1Bm, respectively.
In addition, to construct DGK␥ KN having a nuclear localization signal (NLS), cDNA fragments of DGK␥ KN with BspEI site at the N terminus and XhoI site at the C terminus were produced by a PCR using cDNA for kinase-negative mutant of DGK␥ constructed as described above. The PCR products were first subcloned into a pGEM T-Easy vector (Promega, Madison, WI). After digestion with BspEI and XhoI, the cDNA encoding DGK␥ KN was subcloned into the BspEI and XhoI site in the pECFP-Nuc vector (Clontech). The primers were 5Ј-AATC-CGGGATGAGTGACGGGCAATGG-3Ј and 5Ј-GCTCGAGCGT-CCTTGAACGGCTTTTCCTC-3Ј.
Transfection-Cells (2.0 ϫ 10 4 cells/dish) on glass-bottom dish (Mat-Tek Corp., Ashland, MA) were transfected using 3 l of FuGENE TM 6 transfection reagent (Roche Applied Science) and 1 g of DNA according to the manufacturer's protocol. Transfected cells were cultured at 37°C for ϳ24 h before use.
Immunostaining-CHO-K1 cells transfected cDNA coding FLAG fusion proteins as described above, and those without transfection were fixed for 1 h at room temp with 4% paraformaldehyde, 0.2% picric acid in 0.1 M phosphate buffer and permeabilized with 0.3% TritonX-100 in 0.01 M PBS for 15 min at room temp. Cells were sequentially incubated with 10% normal goat serum, mouse anti-FLAG antibody (Sigma), or anti-DGK␥ antibody, and then Fluorolink Cy3-labeled goat anti-mouse antibody. At each step, transfected cells were washed three times with 0.01 M PBS containing 0.03% Triton X-100 (PBS-T) for 5 min.
For preparation of anti-DGK␥ antibody, an oligopeptide corresponding to amino acids 778 -791 of human DGK␥ (31) was used for antigen. This antibody was purified by antigen-immobilized affinity column. We finally confirmed that the purified DGK␥ antibody has no cross-reactions with pig DGK␣ or rat DGK␤.
Confocal Microscopy-The fluorescence of Cy3 and GFP were observed under confocal laser scanning fluorescent microscopy (Carl Zeiss, Jena, Germany). The GFP fluorescence was monitored at 488 nm argon laser excitation with 515 nm long pass barrier filter. Cy3 fluorescence was monitored at 543 nm HeNe 1 excitation with a 560 -615 nm band pass filter.
Upon quantitating, the subcellular distribution of the proteins fused GFP, which we described below. The cells in which the ratio of GFP fluorescence intensity of cytoplasm and nucleus was under 0.3 were defined as "only in the cytoplasm," and the cells with the ratio of 0.3-0.8 were defined as "abundantly in the cytoplasm." In the case the ratio was over 0.8, the cells were defined as "equally in the cytoplasm and the nucleus." Immunoblotting and Kinase Assay-Plasmids (ϳ32 g) were electroporated into COS-7 cells using a GenePulser (Bio-Rad, 975 microfarads, 220 mV) or transfected into NIH3T3 cells using FuGENE TM 6 transfection reagent. After being cultured, the cells were harvested and centrifuged at 5500 ϫ g for 3 min. The cells were resuspended in homogenizing buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 50 mM Tris-HCl, 200 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% TritonX-100, pH 7.4) and sonicated (UD-210 Tomy, Japan, output 3, 15 s, 2 times).
For immunoblotting, the samples were subjected to 7.5% SDSpolyacrylamide gel electrophoresis, followed by blotting onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Nonspecific binding sites were blocked by incubation with 5% skim milk in PBS-T at 4°C overnight. The membrane was incubated with anti-GFP antibody for 1 h at room temperature. After washing with PBS-T, the membrane was incubated with peroxidase-labeled antirabbit IgG (Jackson, ImmunoResearch Laboratories, West Grove, PA) for 30 min. After three rinses with PBS-T, the immunoreactive bands were visualized using a chemiluminescence detection kit (ECL, Amersham Biosciences).
To determine the kinase activity of DGK␥ and mutants, appropriate volumes of the homogenate samples, which contain comparative amounts of the fusion protein of DGK␥ or mutants assessed by immunoblotting, were subjected to octyl glucoside mixed-micelle assay (14) by subtle modification. 1-Steroyl-2-arachidonoyl-sn-glycerol (Biomol, Plymouth Meeting, PA) was used as substrate. The radioactivity of phosphatidic acid was separated on 20-cm Silica gel 60 TLC plates (Merck) using a chloroform:methanol:acetic acid (65:15:5) solution and detected by using a BAS2500 (Fujix, Tokyo, Japan)

Measurement of the Diameter and Thickness of the Cells Transiently
Expressing Fluorescent Proteins-Plasmids (ϳ5.5 g) encoding GFP-DGK␥, GFP-DGK␥ KN, GFP-DGK␥ ⌬C1B, GFP-DGK␥ KNC1Am, and GFP alone were transfected into CHO-K1 cells by lipofection as described above. Diameters of the respective cells were measured by LSM510 soft ware under confocal microscopy. The thickness of the cells was measured using re-constructed image of three-dimensional sections. Numbers below the graph show the ratio of the cells expressing GFP-DGK␥ in both in the cytoplasm and the nucleus (Type B) to only in the cytoplasm (Type A). The insets in these graphs show the change in the ratio of the cells expressing GFP-DGK␥ both in the cytoplasm and the nucleus to only in the cytoplasm. *, difference of the change in the ratio at each day was significant from that at day 1 (p Ͻ 0.05).

FIGURE 2. Comparison of properties of GFP-DGK␥ and its mutants.
A, constructs of fusion proteins of DGK␥ with GFP and mutants. GFP was fused at the N terminus of DGK␥ (GFP-DGK␥). GFP-DGK␥ ⌬C1A and GFP-DGK␥ ⌬C1B lacked the C1A domain (from 269 to 318 aa) and C1B domain (from 334 to 380 aa), respectively. Cys-285 in the C1A domain or Cys-348 on the C1B domain was replaced to Gly in the GFP-DGK␥ C1Am and GFP-DGK␥ C1Bm, respectively. In GFP-DGK␥ KN, Gly-491 in the ATP binding site was substituted to Asp. GFP-DGK␥ KNC1Am and GFP-DGK␥ KNC1Bm were made by replacing Cys-285 or Cys-348 to Gly in the C1A domain or C1B domain of GFP-DGK␥ KN, respectively. DGK␥-GFP has GFP at the C terminus of DGK␥. B, immunoblot analysis of GFP-DGK␥, DGK␥-GFP, and mutants by anti-GFP antibody. The plasmids were transfected to COS-7 cells by electroporation. The lysates of the transfected cells were subjected to SDS-PAGE and Western blotting as described under "Experimental Procedures." The immunoreactive bands were detected by using anti-GFP antibody. The molecular mass of marker protein is indicated on the left. C, kinase activity of GFP-DGK␥, DGK␥-GFP, and mutants. Comparative amount of the GFP-DGK␥, DGK␥-GFP, and mutants (shown in B) were subjected to the octyl-glucoside method using 1-steroyl-2-arachidonoyl-sn-glycerol as substrate. Reaction products were separated on TLC plate and detected by BAS2500.
Production of Stable Cell Lines-Plasmids (ϳ5.5 g) encoding GFP-DGK␥, GFP-DGK␥ KN, GFP-DGK␥ KNC1Am, and GFP alone were transfected by lipofection using FuGENE TM 6 transfection reagent according to the manufacturer's protocol. Geneticin (0.5 mg/ml) was added to the medium 24 h after the transfection. After being cultured for more than 24 h, the transfected cells were transferred to 96-well plates at 0.5 cell/well for cloning. The positive clone was identified by the fluorescence under confocal microscopy.
Proliferation of Stable Cell Lines-0.6 ϫ 10 6 cells of each stable cell lines were split on three 10-cm dishes (0.2 ϫ 10 6 each). After 24 (day 1), 48 (day 2), and 72 h (day 3), cells were treated with trypsin-EDTA after washed PBS(Ϫ) and collected by centrifugation (1250 ϫ g for 10 min, at 4°C). The harvested cells were resuspended in 1 ml of PBS(Ϫ), stained with 0.4% Trypan blue in PBS(Ϫ), and counted. The doubling time of the lines were calculated from simple regression based on the number of the cells. Difference of correlation coefficients of the regression lines was determined by testing the t value.
Live Imaging of Cell Division of HeLa Cells Expressing CFP-NLS and CFP-DGK␥KN-NLS-Plasmids encoding CFP-NLS and CFP-DGK␥ KN-NLS were transfected into HeLa cells. Images of both phase contrast and CFP fluorescence were taken every 10 min from 24 to 72 h after transfection using fluorescent microscopy, BZ-8000 (Keyence, Osaka, Japan) equipped with cultivation system, INU-KI-F1 (Tokai Hit, Shizuoka, Japan). Based on the images, the doubling time of respective cells, the time period between the first and the second division, was measured.
Flow Cytometry-0.2 ϫ 10 6 cells of each stable cell lines were spread on 10-cm dishes. The cells were synchronized at the beginning of S phase by double thymidine block and release protocol. Briefly, the cells were treated with 10 mM (for GFP stable cells) or 5 mM (for GFP-DGK␥ KN stable cells) thymidine for the first 18 h, followed by an interval of thymidine-free incubation for 10 h, and the second thymidine incubation for 8 h (32). To release the cell cycle, the cells were washed well and cultured in normal medium containing serum. Every hour after the release, cells were treated with trypsin-EDTA and collected in a 1.5-ml tube. Washed by PBS(Ϫ), the cells were fixed by 70% ethanol for 1 h on ice, and treated sequentially by 0.25 mg/ml RNase A and 0.05 mg/ml propidium iodide. Cell cycle analysis was performed by using a FACS-Calibur (BD Biosciences).

RESULTS
Changes in Subcellular Localization of DGK␥-During the cultivation of CHO-K1 cells expressing GFP-DGK␥, we observed different localization of GFP-DGK␥; some cells had GFP-DGK␥ in the nucleus and some did not. Specifically, just after the transfection many of the CHO-K1 cells express GFP-DGK␥ only in the cytoplasm (Fig. 1A, CHO-K1, type A) but 2-3 days later, this enzyme was localized equally in the cytoplasm and nucleus (Fig. 1A, CHO-K1, type B). Here, we considered the possibility that the nuclear localization of GFP-DGK␥ was due to the degradation, because GFP itself is localized in the nucleus and the cytoplasm as shown in below in Fig. 4C. However, no degraded product of GFP-DGK␥ was found even at day 3 by immunoblotting using GFP antibody (Fig. 1B), indicating that the enzyme is transported from the cytoplasm to the nucleus during cultivation. Therefore, we investigated time-dependent changes of GFP-DGK␥ localization. Fig.  1C shows that the number of the CHO-K1 cells expressing GFP-DGK␥ only in the cytoplasm (Type A) decreased, while the number of the cells expressing the fusion protein both in the cytoplasm and the nucleus (Type B) increased with time. Percentage of the type B CHO-K1 cells was 34% at day 1, but it increased to 53% at day 2. The ratio of type B to type A cells changed from 0.826 at day 1 to 2.790 at day 2. This ratio increased further to 3.110 at day 3. We also carried out the same experiment using NIH3T3 cells. In NIH3T3 cells, the percentage of the type A cells and that of the type B cells are similar at day 1, but the type B cells became the major population with time; it increased to 72% at day 2 and 86% at day 3 (Fig. 1C, NIH3T3). Furthermore, we performed this exper-iment using the neuroblastoma cell line, SH-SY5Y, because DGK␥ are abundantly expressed in the brain. In SH-SY5Y cells, DGK␥ also showed different types of the localization (Fig. 1A, SH-SY5Y, type A and type B). Percentage of the type B SH-SY5Y cells was 47% at day 1, and it was 52.2% at day 2 with the change in ratio from 1.395 to 2.373 (Fig. 1C, SH-SY5Y). Finally, the ratio reached 4.183 at day 3. These results indicate that DGK␥ is transported to the nucleus depending on cultivation periods in all cell lines tested.
Mechanism for Nuclear Transportation of DGK␥-To analyze whether enzymatic activity is required for the nuclear transportation of DGK␥, several mutants were generated as shown in Fig. 2A, and correlation between their activities and nuclear transportation was investigated. Immunoblotting using anti-GFP antibody revealed that each mutant had appropriate molecular size (Fig. 2B) and that no significant degraded products were detected. Fig. 2C shows that GFP-DGK␥ had kinase activity, whereas DGK␥ fused GFP at the N terminus (DGK␥-GFP) had no activity as previously reported (28). The mutants having mutations in the C1A or C1B domains (GFP-DGK␥ C1Am and C1Bm) and lacking the C1A or C1B domain (GFP-DGK␥ ⌬C1A and ⌬C1B) showed lower but significant activity than wild-type GFP-DGK␥. Interestingly, mutation in the C1A domain rather than in the C1B domain affected the kinase activity. All the kinase-negative mutants, GFP-DGK␥ KN, KNC1Am, and KNC1Bm, whose Gly-491 in the ATP binding site was replaced to Asp, showed no kinase activity.
Despite no kinase activities, both GFP-DGK␥ KN and DGK␥-GFP were localized in the nucleus as well as the cytoplasm of CHO-K1 cells, but the transportation of GFP-DGK␥ KN into the nucleus was slower than wild type (Fig. 3, GFP-DGK␥ and GFP-DGK␥ KN). On the other hand, GFP-DGK␥ C1Am and C1Bm did not show the increase in the number of cells expressing the mutants equally in the cytoplasm and the nucleus, although they possessed significant kinase activity (Fig. 3, GFP-DGK␥ C1Am and GFP-DGK␥ C1Bm). Similarly, GFP-DGK␥ ⌬C1A, ⌬C1B, KNC1Am, and KNC1Bm did not change their localization (Fig.  3, GFP-DGK␥ ⌬C1A, GFP-DGK␥ ⌬C1B, GFP-DGK␥ KNC1Am, and GFP-DGK␥ KNC1Bm). Table 1 summarizes the kinase activities of the mutants and their transportation to the nucleus. These results show that there is no significant correlation between the kinase activity and the nuclear transportation, suggesting that nuclear transportation of DGK␥ does not require its kinase activity.
On the other hand, as seen in the cases of GFP-DGK␥ C1Am, C1Bm, ⌬C1A, and ⌬C1B and GFP-DGK␥ KNC1Am and KNC1Bm, mutation in the C1A or C1B domains eliminated the nuclear transportation of DGK␥, suggesting that the C1 domain is important for the nuclear transportation of DGK␥ and contains a nuclear transport signal. To confirm the function of C1 domain as a nuclear transport signal and

TABLE 1 The activity of GFP-DGK␥ , DGK␥ -GFP, and mutants and their nuclear localization in CHO-K1 cells
The number of "ϩ" symbols in the box of the activity of DGK represents the strength of kinase activity. In the box of the localization in the nucleus, "ϩ" represents the exogenous proteins that are localized in the nucleus, and "Ϫ" represents exogenous proteins that are not in the nucleus.

The activity of DGK Localization in the nucleus
Ϫ Ϫ  (Fig. 4A) and investigated their localization using FLAG antibody. The entire C1 domain was dominantly localized in the nucleus but the other fragments did not show nuclear localization (Fig. 4B). Similarly, GFP-tagged C1 domain was localized specifically in the nucleus, whereas GFP alone was seen throughout the cells (Fig. 4C). These results represent that the entire C1 domain is necessary to act as a nuclear localization signal at least in DGK␥.   (Fig. 5, left side), suggesting the importance of DGK␥ activity in cell shape. However, it is not clear whether the difference in the cell size was due to the lack of DGK␥ the activity in the nucleus or in the cytoplasm because GFP-DGK␥ KN expressed both in the cytoplasm and the nucleus. We, therefore, further examined the size of cells expressing DGK mutants only in the cytoplasm to examine an effect of cytoplasmic DGK activity on the cell size. We used the cells expressing GFP-DGK␥ KNC1Am, because the mutants has no kinase activity and localizes only in the cytoplasm. On the other hand, the cells expressing GFP-DGK␥ ⌬C1B were used as the cells with cytoplasmic kinase activity (Figs. 2C and 3). No differences were found in the size of cells expressing GFP-DGK␥ KNC1Am and GFP-DGK␥ ⌬C1B; the size of cells expressing GFP-DGK␥ ⌬C1B and KNC1Am were similar to that of GFPor GFP-DGK␥-expressing cells (Fig. 5, left side). Furthermore, we measured the thickness of the CHO-K1 cells transiently expressing these proteins to investigate whether overexpression of DGK␥KN flattened the cells and/or resulted in increase in cell volume. As shown in Fig. 5, the cells expressing GFP-DGK␥ KN were slightly flatter than those of the cells expressing other proteins. In addition, volume of the GFP-DGK␥ KN cells was ϳ1.36-fold; the average of the diameter and thickness of GFP-DGK␥ KN cells were 34.7 m and 7.98 m, whereas those of the other cells were 28.86 m and 8.46 m, respectively. These results indicated that the overexpression of DGK␥ KN affected the cell shape and volume. To confirm this, we further made stable CHO-K1 cell lines of GFP, GFP-DGK␥, GFP-DGK␥ KN, and GFP-DGK␥ KNC1Am and compared the size of the cells. The stable cells expressing GFP-DGK␥ KN in the nucleus and the cytoplasm were remarkably bigger than the others (Table 2). Importantly, the size of cells expressing GFP, GFP-DGK␥, and GFP-DGK␥ KNC1Am were almost same. These results indicate that dominant negative effect on the nuclear DGK␥, but not cytoplasmic DGK␥, influences the size and volume of cells.

Nuclear Transportation and Function of DGK␥
Next, to elucidate the mechanism of DGK␥ KN-induced enlargement of cells, we compared proliferation of the stable cell lines (Fig. 6). Fig. 6 shows that the DGK␥KN cells stated the division more slowly than the others and that doubling time of GFP-DGK␥KN stable cells was 19.1 h, which was calculated from its regression line. On the other hand, those of GFP, GFP-DGK␥, and GFP-DGK␥KNC1Am cells were ϳ15.4, 15.8, and 15.8 h. These results indicated that the proliferation rate of GFP-DGK␥ KN was slightly slower than others. To directly investigate inhibitory effect of nuclear DGK␥ KN on the proliferation, we constructed CFP-DGK␥ KN having nuclear localization signal (CFP-DGK␥ KN-NLS) and CFP-NLS as control and compared their proliferation rate.
The cell expressing CFP-DGK␥ KN-NLS had a tendency to die, compared with control cells. Doubling time of the cells transiently expressing CFP-DGK␥ KN-NLS was 17.36 Ϯ 1.99 h (n ϭ 25), whereas that of control cells was 15.58 Ϯ 1.69 h (n ϭ 50). These results demonstrated that nuclear GFP-DGK␥ KN causes the disorder of cell cycle.
Therefore, we measured cell cycle of GFP-DGK␥ KN by flow cytometry and compared it with control cell lines expressing GFP alone and GFP-DGK␥ KNC1Am, the latter of which is inactive and cytosolic. At 24 h after the release from serum starvation, the profile of GFP-DGK␥ KN stable cells was different from those of GFP and GFP-DGK␥ KNC1Am stable cells, indicating that nuclear, but not cytosolic DGK␥ KN affected the cell cycle. Then, we analyzed the cell cycle more precisely using GFP and GFP-DGK␥ KN stable cells synchronized at the beginning of S phase by the double thymidine block method (32). Prior to the experiment, we determined proper concentration of thymidine for synchronizing the cells (supplemental Fig. S1). In the case of GFP-DGK␥ KN stable cell lines, treatment with 2.5 mM thymidine resulted in partial G 1 arrest, and 5 mM thymidine exerted a much clearer effect. For GFP stable cell lines, G 1 arrest was not induced at 5 mM, and 10 mM was needed for complete G 1 arrest. From these results, we decided to perform double thymidine block at 10 mM for GFP stable cells and 5 mM for GFP-DGK␥ KN cells for cell-cycle analysis.
At 4 h after washing out thymidine, ϳ37% of the control cells transited to G 2 /M phase, whereas the cells expressing GFP-DGK␥ KN in G 2 /M phase were only 17% (Fig. 7). ϳ70% of the control cells were in G 2 /M phase at 5 h, but it took 6 -7 h for the population of GFP-DGK␥ KN in G 2 /M phase to reach maximum. The GFP stable cells almost returned to G 1 phase at 7 h, and the size of population of them in G 1 phase reached 60% at 8 h, the biggest value through this experiment. On the other hand, 7 h after the release, ϳ51% of GFP-DGK␥ KN stable cells still remained in G 2 /M phase, and only 14% cells were in G 1 phase. Finally, a major population of GFP-DGK␥ KN transited to G 1 phase at 9 h; it was delayed by 2 h compared with the GFP stable cells. These results clearly reveal that the S phase of GFP-DGK␥ KN was extended, suggesting that the extension causes enlargement of the cell.
The disorder of cell cycle by nuclear expression of DGK␥ KN suggested that alteration of the cell cycle affects the intracellular localization of DGK␥. Therefore, we observed the localization of GFP-DGK␥ in the stable cells after serum starvation for 24 h. Under control conditions, the percentage of the stable cells expressing GFP-DGK␥ both in the cytoplasm and the nucleus was 34% and that expressing only in the cytoplasm was 51% of all cells (Fig. 8A, control). The number of the cells expressing GFP-DGK␥ both in the cytoplasm and the nucleus (type B) increased from 35% to 54% by serum starvation. In contrast, the number of the cells expressing the fusion protein only in the cytoplasm (type A) decreased from 51% to 34%. The ratio of type B to type A increased from 0.692 to 1.557. These results indicate that GFP-DGK␥ is transported from the cytoplasm to the nucleus under serum-starved conditions. However, these experiments were carried out under artificial conditions using exogenously expressed GFP-DGK␥. Therefore, to confirm nuclear transportation of endogenous DGK␥, we performed immunostaining using anti-DGK␥ antibody. In many control cells, endogenous DGK␥ was localized in the cytoplasm but not in the nucleus (Fig. 8B,  control). After 24-h serum starvation, DGK␥ localized in the nucleus as much as in the cytoplasm (Fig. 8, serum starvation), indicating that, as well as GFP-DGK␥, endogenous DGK␥ can be transported to the nucleus, and the nuclear transportation is induced by serum starvation. These results, together with the inhibitory effect of DGK␥KN on cell cycle, strongly suggest an important physiological function of nuclear DGK␥ in the cell cycle regulation.

TABLE 2 Sizes of the stable cell lines of GFP, GFP-DGK␥ , GFP-DGK␥ KN, and GFP-DGK␥ KNC1Am
The cells were spread onto grass bottom dishes. After being cultured for 72 h, the cells were fixed, and the size of each cell was measured using LSM510 software. Averages of sizes of the 100 cells are shown. In the case of GFP, GFP-DGK␥ , and GFP-DGK␥ KN, the cells expressed both in the cytoplasm and the nucleus were chosen. On the other hand, the cells expressed only in the cytoplasm were selected in the case of GFP-DGK␥ KNC1Am.

DISCUSSION
In this report, we showed for the first time that DGK␥ is transported from the cytoplasm to the nucleus (Fig. 1). The nuclear transportation of DGK␥ was independent of kinase activity of DGK␥ ( Fig. 3 and Table 1), but the mutant lacking kinase activity (DGK␥ KN) showed much slower nuclear transportation than that of wild type. The mutation eliminating kinase activity of GFP-DGK␥ C1Am or GFP-DGK␥ C1Bm made a remark-able decrease in the number of the cells expressing equally in the cytoplasm and the nucleus (GFP-DGK␥ KNC1Am versus GFP-DGK␥ C1Am or GFP-DGK␥ KNC1Bm versus GFP-DGK␥ C1Bm in Fig. 3). These results suggest that the kinase activity, although not essential, may have some roles in the nuclear transportation of DGK␥.
Instead of kinase activity, the C1 domain of DGK␥ was essential for the nuclear transportation of DGK␥. Namely, none of the C1 domain mutants (GFP-DGK␥ C1Am and C1Bm or GFP-DGK␥ ⌬C1A and ⌬C1B) showed transportation from the cytoplasm to the nucleus ( Fig. 3 and Table 1). In addition, FLAG and GFP tagging the entire C1 domain were dominantly localized only in the nucleus, and the entire C1 domain was necessary for the nuclear localization (Fig. 4). These results suggest that, at least in DGK␥, the entire C1 domain acts as a nuclear localization signal.
However, the C1 domain of DGK␥ doesn't possess a well known NLS, and it is not clear how it acts as a nuclear transport signal. It has been already reported that C1 domains of PKC, DGK, and chimaerin can bind to some lipids (29,33,34). Thus, lipids including DG may be involved in the nuclear transportation of DGK␥. In fact, the mutations in the C1 domain, which are predicted to weaken or abolish their lipid binding based on the fact that corresponding mutations in the ␦PKC C1 domain abrogate the phorbol 12,13-dibutyrate binding (35), inhibited the nuclear transportation. Alternatively, carrier proteins such as importin (36,37) may participate in the nuclear transportation by associating with C1 domain, because the C1 domain of DGK can interact with some proteins (38). The identification of lipid(s) or protein(s) binding to DGK␥ C1 domain would be helpful to understand the mechanism of C1 domain as NLS. Furthermore, it is interesting to study whether other C1 domains in other DGKs and PKCs are important for nuclear localization or not.
In addition to the nuclear transportation mechanism, DGK␥ seems to have export mechanism. Although the C1 domain of DGK␥ was localized dominantly in the nucleus (Fig. 4, B and C), full-length DGK␥ was expressed equally in the cytoplasm and the nucleus (Figs. 1 and 3). We could not find the cells that expressed DGK␥ in the nucleus dominant over the cytoplasm. In addition, the photobleaching of cytoplasmic fluorescence of the cells expressing GFP-DGK␥ caused a rapid decrease in the nuclear fluorescence within 5-10 min, and then the cytoplasmic fluorescence reached a level equal to that of the nucleus (data not shown). These results suggest that DGK has not only nuclear transport mechanism but also export mechanism and that the DGK level in the nucleus is maintained as much as that in the cytoplasm. Probably, the shuttling of DGK␥ between the cytoplasm and the nucleus is regulated through the C1 domain and other region(s).
We also investigated physiological function of nuclear DGK␥ by expressing kinase negative DGK␥ in the nucleus, which is expected to inhibit the endogenous nuclear DGK␥ by dominant-negative effects. The cells expressing GFP-DGK␥ KN were larger in the size ( Fig. 5 and Table 2), proliferated slowly (Fig. 6), and their S phase was extended (Fig. 7), suggesting that enlargement of cell size is induced by protein synthesis during extended S phase. In addition, serum starvation induced nuclear transportation of endogenous DGK␥ and GFP-DGK␥ stably expressing in the CHO-K1 cells (Fig. 8). These results strongly implicate that DGK␥ in the nucleus is involved in regulation of cell cycle.
Involvement of DGK in cell-cycle regulation is supported by several reports as follows. First, inhibition of DGK activity prevents transition from G 1 to S phase (39). Second, the involvement of other DGK subtypes such as DGK in the cell cycle has also been reported, although there are some differences; COS-7 cells expressing DGK showed the increase in the size of G 1 cell population and decrease in that of G 2 /M cell population but no effects of DGK KN on the cell cycle were detected (21). The differences may be due to subtype specificity and/or different cell types used.
Both DGK␥ and DGK are abundantly expressed in the brain (40), although it is unclear about their functions as a regulator of the cell cycle in the central nervous system. However, expression of DGK␥ has been recently reported in HL-60, U937, and NIH3T3 cells (41)(42)(43). We also confirmed the expression of DGK␥ in various tissues, including kidney, muscle, and other tissues, in addition to brain (supplemental Fig. S2). Therefore, at least in these cells and tissues, it is expected that nuclear DGK␥ is involved in cell-cycle regulation. In fact, regulation of differentiation by DGK␥ in HL-60 cells has been suggested (41,43).
However, it still remains to be solved how DGK can regulate the cell cycle. The amount of nuclear phosphatidylinositols, including phosphatidylinositol 4,5-bisphosphate, fluctuates during the cell cycle (22), and the DG mass in the nucleus increases in G 2 /M phase (27). Furthermore, ␤IIPKC translocates into the nucleus and phosphorylates lamin B Moreover, 100 cells were observed, and the number of the cells expressing GFP-DGK␥ only in the cytoplasm (shaded bars), abundantly in the cytoplasm (Ⅺ), and equally in the cytoplasm and the nucleus (f) were counted. Same experiment was independently performed three times and the averages Ϯ S.E. are plotted as percentage. Numbers below the graph show the ratio of the cells expressing GFP-DGK␥ in both the cytoplasm and the nucleus to only in the cytoplasm. B, the localization of endogenous DGK␥ in the intact cells under serum starvation conditions. CHO-K1 cells were spread onto glass bottom dishes and cultured overnight. After being cultured for an additional 24 h in the presence (control) and absence of serum (serum starvation), the cells were stained with anti-DGK␥ antibody. As negative control, normal rabbit serum was used instead of anti-DGK␥ antibody.
in the G 2 /M phase (44). These reports suggest that the DG derived from phosphatidylinositol 4,5-bisphosphate recruits PKC to regulate G 2 /M phase. In other words, regulation of nuclear DG mass is important for cell-cycle regulation, and DGK might be involved in the control of nuclear DG. Alternatively, PA produced by DGK may have some function in cell-cycle regulation. In fact, the amount of PA in the nucleus also changes by the stimulation of ␣-thrombin, which has a mitogenic effect (45). Accordingly, the mechanism of cell-cycle regulation by DGK␥ is an important issue to be solved next.
In conclusion, the present results indicate that DGK␥ is transported from the cytoplasm to the nucleus via its C1 domain and suggest that DGK␥ is involved in cell-cycle regulation.