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

Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp. 6152–6164, March 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Nuclear Transportation of Diacylglycerol Kinase  and Its □ S Possible Function in the Nucleus Received for publication, September 7, 2005, and in revised form, December 27, 2005 Published, JBC Papers in Press, December 30, 2005, DOI 10.1074/jbc.M509873200 ‡ ‡1 ‡ ‡ ‡ Takehiro Matsubara , Yasuhito Shirai , Kei Miyasaka , Takuya Murakami , Yasuto Yamaguchi , ‡ § § § ¶ ¶ Takehiko Ueyama , Masahiro Kai , Fumio Sakane , Hideo Kanoh , Toshiaki Hashimoto , Shinji Kamada , ¶ ‡2 Ushio Kikkawa , and Naoaki Saito ‡ ¶ From the Laboratory of Molecular Pharmacology and Laboratory of Biochemistry, Biosignal Research Center, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501 and the Department of Biochemistry, Sapporo Medical University School of Medicine, West-17, South-1, Chuo-Ku, Sapporo 060-8556, Japan Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to consists of at least nine subtypes (2). Although all DGKs have cysteine- phosphatidic acid, and both lipids are known to play important rich repeats similar to the C1A and C1B domains of PKCs in the N roles in lipid signal transduction. Thereby, DGKs are considered to terminus and a catalytic domain in the C terminus, they are divided into be a one of the key players in lipid signaling, but its physiological five groups on the primary structure of these DGKs. Type I DGKs, function remains to be solved. In an effort to investigate one of nine including DGK,-, and -, have EF-hand motifs and two cysteine-rich subtypes, we found that DGK came to be localized in the nucleus regions (C1 domain) in the regulatory domain (12, 13), whereas Type II with time in all cell lines tested while seen only in the cytoplasm at DGKs, DGK and -, have a pleckstrin homology domain instead of the the early stage of culture, indicating that DGK is transported from EF-hand motif in addition to the C1 domain (14, 15). The separated the cytoplasm to the nucleus. The nuclear transportation of DGK catalytic domains of DGK and - are characteristic of Type II DGK. didn’t necessarily need DGK activity, but its C1 domain was indis- Type III, DGK, has only one C1 domain in the regulatory domain (16). pensable, suggesting that the C1 domain of DGK acts as a nuclear Myristoylated alanine-rich protein kinase C substrate phosphorylation transport signal. Furthermore, to address the function of DGK in site-like region and four ankyrin repeats are unique domains in Type IV the nucleus, we produced stable cell lines of wild-type DGK and DGK (17, 18). The final group, type V, includes DGK, which has three mutants, including kinase negative, and investigated their cell size, cysteine-rich regions and a pleckstrin homology domain with overlap- growth rate, and cell cycle. The cells expressing the kinase-negative ping Ras-associating domain (19). The DGKs are thought to be involved mutant of DGK were larger in size and showed slower growth rate, in development, differentiation, construction of neural network and and the S phase of the cells was extended. These findings implicate immunity, etc. However, subtype specific function and regulation that nuclear DGK regulates cell cycle. mechanisms of DGKs are not clear. Several groups have reported many different localization and trans- location of DGKs, possibly contributing to their subtype-specific func- Diacylglycerol (DG) is a second messenger regulating various cellu- tions. In an effort to elucidate the function of DGK, we unexpectedly lar responses (1, 2). One of the important roles of DG is an activating of found that GFP-fused DGK (GFP-DGK) became localized in the protein kinase C (PKC) (1, 3, 4). DG is physiologically produced as a result of the signal-induced hydrolysis of phosphatidylinositol by phos- nucleus as well as the cytoplasm a few days after transfection but was pholipase C. The generated DG is phosphorylated to phosphatidic acid localized mainly in the cytoplasm just after expressed in CHO-K1 cells. by diacylglycerol kinase (DGK) or metabolized by DG lipase (2, 5, 6). Although nuclear transportation of DGK has never been reported, Thus, DGK is an important enzyme to inactivate PKC by reducing the expression of DGK and DGK in the nucleus has been already DG level, contributing to regulating of the cellular response. In addition, described (20, 21). In addition, DGK is thought to be involved in the phosphatidic acid itself activates PKC (7), phosphatidylinositol regulation of cell cycle (21). These findings, together with the facts that 4-phosphate 5-kinase (8, 9), and mammalian target of rapamycin (10), phosphatidylinositol turnover exists within the nucleus and DG may be and modulates Ras GTPase-activating protein (11). involved in the regulation of cell cycle (22–27), suggest that DGK has Molecular cloning studies revealed that mammalian DGK family some physiological function in the nucleus. However, mechanism of the nuclear transportation and physiological functions of DGK are *This work was supported by grants from the 21st Century Center of Excellence Program unknown. We, therefore, investigated molecular mechanism and phys- of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from iological significance of nuclear transportation of DGK. 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 EXPERIMENTAL PROCEDURES the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S Materials—CHO-K1 cells were donated from Dr. M. Nishijima The on-line version of this article (available at http://www.jbc.org) contains supple- mental Figs. S1 and S2. (National Institute of Health, Tokyo, Japan). COS-7 cells, SH-SY5Y To whom correspondence may be addressed. Tel.: 81-78-803-5961; Fax: 81-78-803- cells, and HeLa cells were purchased from the RIKEN Cell Bank. Fetal 5971; E-mail: [email protected]. To whom correspondence may be addressed. Tel.: 81-78-803-5961; Fax: 81-78-803- bovine serum, RNase A, and anti-FLAG M2 monoclonal antibody were 5971; E-mail: [email protected]. obtained from Sigma. FuGENE 6 Transfection reagent was obtained The abbreviations used are: DG, diacylglycerol; PKC, protein kinase C; DGK, diacylglyc- erol kinase; CHO, Chinese hamster overly; C1, conserved region 1; C1A, first half of C1 from Roche Applied Science. Propidium iodide was obtained from domain; C1B, second half of C1 domain; KN, kinase negative; COS, African green Wako (Osaka, Japan). FluoroLink Cy3-labeled goat anti-mouse IgG was monkey kidney; GFP, green fluorescent protein; NLS, nuclear localization signal; PBS, phosphate-buffered saline; aa, amino acid(s). purchased from Amersham Biosciences. We produced anti-GFP anti- 6152 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 This is an Open Access article under the CC BY license. Nuclear Transportation and Function of DGK 4 32 body ourselves. [- P]ATP was obtained from ICN Biomedicals CGAATTCATGGTGAAAACATACTCC-3 and 5-TGAGATCTG- (Irvine, CA). A TLC plate was obtained from Merck (Darmstadt, Ger- CACGCTTGCACAAT-3. The primers for C1A domain (805–954 bp) many). Geneticin was purchased from Invitrogen. were 5-TTGAATTCATGCCACGCCTGGACCCCTGA-3 and 5-T- Cell Culture—CHO-K1 cells were cultured in Ham’s F-12 medium TAGATCTCTGCATCACCTCACC-3. The primers for Hinge (Nacalaitesque, Japan). COS-7 and NIH3T3 cells were cultured in Dul- (955–990 bp) were 5-CCGAATTCATGGTGAAAACATACTCC-3 becco’s modified Eagles’ medium (Nacalaitesque, Japan). SH-SY5Y cells and 5-TTAGATCTCTGCATCACCTCACC-3. The primers for C1B were cultured in Dulbecco’s modified Eagles’ medium/Ham’s F-12 domain (1000–1140 bp) were 5-TTGAATTCATGCACGCGTGGG- medium (1:1) (Invitrogen). All cells were cultured at 37 °C in humidified TGGAA-3 and 5-TGAGATCTGCACGCTTGTCGACATA-3. atmosphere containing 5% CO . All media contained 25 mM glucose, Transfection—Cells (2.0 10 cells/dish) on glass-bottom dish (Mat- TM and all were buffered with 44 mM NaHCO and supplemented with 10% Tek Corp., Ashland, MA) were transfected using 3 l of FuGENE 6 fetal bovine serum (Sigma), penicillin (100 units/ml), and streptomycin transfection reagent (Roche Applied Science) and 1 g of DNA accord- (100 g/ml) (Invitrogen). The fetal bovine serum used was not heat- ing to the manufacturer’s protocol. Transfected cells were cultured at inactivated. The media for SH-SY5Y cells was added with 1% GlutaMA- 37 °C for 24 h before use. TM X -I Supplement (Invitrogen). Immunostaining—CHO-K1 cells transfected cDNA coding FLAG Constructs of Plasmids Encoding DGK-fused GFP and Mutants— fusion proteins as described above, and those without transfection were The constructs encoding DGK having GFP at its N terminus (GFP- fixed for1hat room temp with 4% paraformaldehyde, 0.2% picric acid in DGK) or at C terminus (DGK-GFP) and mutants having substitution 0.1 M phosphate buffer and permeabilized with 0.3% TritonX-100 in of Cys-285 to Gly in the C1A domain (GFP-DGK C1Am) or Cys-348 to 0.01 M PBS for 15 min at room temp. Cells were sequentially incubated Gly in the C1B domain (GFP-DGK C1Bm) were previously described with 10% normal goat serum, mouse anti-FLAG antibody (Sigma), or (28). The constructs encoding the mutants lacking the C1A or C1B anti-DGK antibody, and then Fluorolink Cy3-labeled goat anti-mouse domain (GFP-DGK C1A or GFP-DGK C1B) were also described antibody. At each step, transfected cells were washed three times with previously (29). Kinase negative mutant of DGK (GFP-DGK KN) was 0.01 M PBS containing 0.03% Triton X-100 (PBS-T) for 5 min. produced by substitution of Gly-491 to Asp. Briefly, using the plasmid For preparation of anti-DGK antibody, an oligopeptide correspond- encoding GFP-DGK, site-directed mutagenesis was performed ing to amino acids 778–791 of human DGK (31) was used for antigen. according to the manufacturer’s recommended protocol with an ExSite This antibody was purified by antigen-immobilized affinity column. We PCR-based site-directed mutagenesis kit (Stratagene). The primers finally confirmed that the purified DGK antibody has no cross-reac- were 5-GCTGGATCCTGGATTGCTTTGACAAGG-3 and 5-CA- tions with pig DGK or rat DGK. ACTGTGTCATCTCCGCCACAGGCAA-3. The mutation was con- Confocal Microscopy—The fluorescence of Cy3 and GFP were firmed by verifying its sequence. Furthermore, to make kinase-negative observed under confocal laser scanning fluorescent microscopy (Carl mutants having substitution of Cys-285 or Cys-348 to Gly (GFP-DGK Zeiss, Jena, Germany). The GFP fluorescence was monitored at 488 nm KNC1Am and GGFP-DGK KNC1Bm), the cDNA of DGK KN was argon laser excitation with 515 nm long pass barrier filter. Cy3 fluores- digested with SalI and SmaI and then subcloned into the SalI and SmaI cence was monitored at 543 nm HeNe 1 excitation with a 560–615 nm site in the plasmid encoding GFP-DGK C1Am and GFP-DGK C1Bm, band pass filter. respectively. Upon quantitating, the subcellular distribution of the proteins fused In addition, to construct DGK KN having a nuclear localization GFP, which we described below. The cells in which the ratio of GFP fluo- signal (NLS), cDNA fragments of DGK KN with BspEI site at the N rescence intensity of cytoplasm and nucleus was under 0.3 were defined as terminus and XhoI site at the C terminus were produced by a PCR using “only in the cytoplasm,” and the cells with the ratio of 0.3–0.8 were defined cDNA for kinase-negative mutant of DGK constructed as described as “abundantly in the cytoplasm.” In the case the ratio was over 0.8, the cells above. The PCR products were first subcloned into a pGEM T-Easy were defined as “equally in the cytoplasm and the nucleus.” vector (Promega, Madison, WI). After digestion with BspEI and XhoI, Immunoblotting and Kinase Assay—Plasmids (32 g) were electro- the cDNA encoding DGK KN was subcloned into the BspEI and XhoI porated into COS-7 cells using a GenePulser (Bio-Rad, 975 microfarads, site in the pECFP-Nuc vector (Clontech). The primers were 5-AATC- TM 220 mV) or transfected into NIH3T3 cells using FuGENE 6 transfection CGGGATGAGTGACGGGCAATGG-3 and 5-GCTCGAGCGT- reagent. After being cultured, the cells were harvested and centrifuged at CCTTGAACGGCTTTTCCTC-3. 5500  g for 3 min. The cells were resuspended in homogenizing buffer Constructs of the Plasmids Encoding C1 Domain Fragments of (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 50 mM Tris-HCl, 200 g/ml DGK—cDNA fragments of C1 domain with EcoRI site in the N termi- leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% TritonX-100, pH 7.4) nus and BamHI site in the C terminus were produced by a PCR using and sonicated (UD-210 Tomy, Japan, output 3, 15 s, 2 times). cDNA for rat DGK as the template (30). The PCR products were first For immunoblotting, the samples were subjected to 7.5% SDS- subcloned into a pGEM T-Easy vector (Promega). After digestion with polyacrylamide gel electrophoresis, followed by blotting onto a poly- EcoRI and BamHI, the cDNA-encoding C1 domain was subcloned into vinylidene difluoride membrane (Millipore, Bedford, MA). Nonspe- TM the EcoRI and BglII sites in the p3xFLAG-CMV -14 expression vector cific binding sites were blocked by incubation with 5% skim milk in (Sigma) or pEGFPC2 (Clontech). The primers for C1 domain (805– PBS-T at 4 °C overnight. The membrane was incubated with anti- 1140 bp) were 5-TTGAATTCATGCACGCCTGGACCCGA-3 and GFP antibody for 1 h at room temperature. After washing with 5-TGAGATCTGCACGCTGTCGACCAAT-3. The primers for C1A PBS-T, the membrane was incubated with peroxidase-labeled anti- domain plus Hinge (805–999 bp) were 5-TTGAATTCATGCACGG- rabbit IgG (Jackson, ImmunoResearch Laboratories, West Grove, CCTGGACCCTG-3 and 5-TTAGATCTCTGCATCACCTCACC- PA) for 30 min. After three rinses with PBS-T, the immunoreactive 3. The primers for Hinge plus C1B domain (955–1140 bp) were 5-C- bands were visualized using a chemiluminescence detection kit (ECL, Amersham Biosciences). To determine the kinase activity of DGK and mutants, appropriate Y. Yamaguchi, Y. Shirai, T. Matsubara, N. Ohshiro, K. Yoshino, K. Yonezawa, Y. Ono, and N. Saito, manuscript in preparation. volumes of the homogenate samples, which contain comparative MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6153 Nuclear Transportation and Function of DGK FIGURE 1. Changes in subcellular localization of GFP-DGK in CHO-K1, NIH3T3, and SH-SY5Y cells. A, different type of DGK localization in CHO-K1 cells, NIH3T3 cells, and SH-SY5Y cells. Plas- mids of GFP-DGK were transfected by lipofection using FuGENE in respective cell lines. 24 h after transfection, some cells express GFP-DGK only in cytoplasm (Type A), and the fusion proteins are localized equally in the cytoplasm and the nucleus (Type B). B, immunoblot analysis of GFP-DGK at days 1 and 3 after transfection by anti-GFP anti- body. The plasmids were transfected to NIH3T3 cells by lipofection. The cells were harvested 1 or 3 days after transfection. The lysates of the trans- fected cells were subjected to SDS-PAGE and Western blotting as described under “Experimen- tal Procedures.” The immunoreactive bands were detected by using anti-GFP antibody. The molec- ular mass of marker protein is indicated on the left. C, time-dependent change of DGK localization in CHO-K1, NIH3T3, and SH-SY5Y cells. More than 100 cells were observed at 24 h (day 1), 48 h (day 2), and 72 h (day 3) after transfection, respectively, and the number of the cells expressing GFP-DGK only in the cytoplasm (shaded bars), abundantly in the cytoplasm () and equally in the cytoplasm and the nucleus (f) were counted. Same experi- ment was independently performed three times, and the averages S.E. are plotted as percentage. Numbers below the graph show the ratio of the cells expressing GFP-DGK in both in the cyto- plasm and the nucleus (Type B) to only in the cyto- plasm (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 day1(p  0.05). amounts of the fusion protein of DGK or mutants assessed by immu- Measurement of the Diameter and Thickness of the Cells Transiently noblotting, were subjected to octyl glucoside mixed-micelle assay (14) Expressing Fluorescent Proteins—Plasmids (5.5 g) encoding GFP- by subtle modification. 1-Steroyl-2-arachidonoyl-sn-glycerol (Biomol, DGK, GFP-DGK KN, GFP-DGK C1B, GFP-DGK KNC1Am, and Plymouth Meeting, PA) was used as substrate. The radioactivity of GFP alone were transfected into CHO-K1 cells by lipofection as described phosphatidic acid was separated on 20-cm Silica gel 60 TLC plates above. Diameters of the respective cells were measured by LSM510 soft (Merck) using a chloroform:methanol:acetic acid (65:15:5) solution and ware under confocal microscopy. The thickness of the cells was measured detected by using a BAS2500 (Fujix, Tokyo, Japan) using re-constructed image of three-dimensional sections. 6154 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK 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. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6155 Nuclear Transportation and Function of DGK FIGURE 3. Changes in subcellular localization of DGK mutants in CHO-K1 cells. The number of the cells expressing GFP-DGK KN, DGK-GFP, GFP-DGK C1Am, GFP-DGK C1Bm, GFP-DGK C1A, GFP-DGK C1B, GFP-DGK KNC1Am, and GFP-DGK KNC1Bm only in the cytoplasm (shaded bars), abundantly in the cytoplasm () and equally in the cytoplasm and the nucleus (f), were counted at each time point and shown as percentage. Numbers below the graph show the ratio of numbers of the cells expressing each mutants or DGK-GFP both in the cytoplasm and the nucleus to only in the cytoplasm. The insets in these graphs show the change in the ratio of the cells expressing each mutants or DGK-GFP 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  0.05). Production of Stable Cell Lines—Plasmids (5.5 g) encoding GFP- according to the manufacturer’s protocol. Geneticin (0.5 mg/ml) was DGK, GFP-DGK KN, GFP-DGK KNC1Am, and GFP alone were added to the medium 24 h after the transfection. After being cultured TM transfected by lipofection using FuGENE 6 transfection reagent for more than 24 h, the transfected cells were transferred to 96-well 6156 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK plates at 0.5 cell/well for cloning. The positive clone was identified by TABLE 1 the fluorescence under confocal microscopy. The activity of GFP-DGK , DGK -GFP, and mutants and their nuclear localization in CHO-K1 cells Proliferation of Stable Cell Lines—0.6  10 cells of each stable cell 6 The number of “” symbols in the box of the activity of DGK represents the strength lines were split on three 10-cm dishes (0.2  10 each). After 24 (day 1), of kinase activity. In the box of the localization in the nucleus, “” represents the 48 (day 2), and 72 h (day 3), cells were treated with trypsin-EDTA after exogenous proteins that are localized in the nucleus, and “” represents exogenous proteins that are not in the nucleus. washed PBS() and collected by centrifugation (1250  g for 10 min, at The activity of Localization in the 4 °C). The harvested cells were resuspended in 1 ml of PBS(), stained DGK nucleus with 0.4% Trypan blue in PBS(), and counted. The doubling time of GFP-DGK the lines were calculated from simple regression based on the number of GFP-DGK KN the cells. Difference of correlation coefficients of the regression lines DGK-GFP GFP-DGK C1Am was determined by testing the t value. GFP-DGK C1Bm Live Imaging of Cell Division of HeLa Cells Expressing CFP-NLS and GFP-DGK C1A GFP-DGK C1B CFP-DGKKN-NLS—Plasmids encoding CFP-NLS and CFP-DGK GFP-DGK KNC1Am KN-NLS were transfected into HeLa cells. Images of both phase con- GFP-DGK KNC1Bm trast and CFP fluorescence were taken every 10 min from 24 to 72 h after transfection using fluorescent microscopy, BZ-8000 (Keyence, Osaka, iment using the neuroblastoma cell line, SH-SY5Y, because DGK are Japan) equipped with cultivation system, INU-KI-F1 (Tokai Hit, Shi- abundantly expressed in the brain. In SH-SY5Y cells, DGK also zuoka, Japan). Based on the images, the doubling time of respective cells, showed different types of the localization (Fig. 1A, SH-SY5Y, type A and the time period between the first and the second division, was type B). Percentage of the type B SH-SY5Y cells was 47% at day 1, and it measured. was 52.2% at day 2 with the change in ratio from 1.395 to 2.373 (Fig. 1C, Flow Cytometry—0.2  10 cells of each stable cell lines were spread SH-SY5Y). Finally, the ratio reached 4.183 at day 3. These results indi- on 10-cm dishes. The cells were synchronized at the beginning of S cate that DGK is transported to the nucleus depending on cultivation phase by double thymidine block and release protocol. Briefly, the cells periods in all cell lines tested. were treated with 10 mM (for GFP stable cells) or 5 mM (for GFP-DGK Mechanism for Nuclear Transportation of DGK—To analyze KN stable cells) thymidine for the first 18 h, followed by an interval of whether enzymatic activity is required for the nuclear transportation of thymidine-free incubation for 10 h, and the second thymidine incuba- DGK, several mutants were generated as shown in Fig. 2A, and corre- tion for 8 h (32). To release the cell cycle, the cells were washed well and lation between their activities and nuclear transportation was investi- cultured in normal medium containing serum. Every hour after the gated. Immunoblotting using anti-GFP antibody revealed that each release, cells were treated with trypsin-EDTA and collected in a 1.5-ml mutant had appropriate molecular size (Fig. 2B) and that no significant tube. Washed by PBS(), the cells were fixed by 70% ethanol for1hon degraded products were detected. Fig. 2C shows that GFP-DGK had ice, and treated sequentially by 0.25 mg/ml RNase A and 0.05 mg/ml kinase activity, whereas DGK fused GFP at the N terminus (DGK- propidium iodide. Cell cycle analysis was performed by using a FACS- GFP) had no activity as previously reported (28). The mutants having Calibur (BD Biosciences). mutations in the C1A or C1B domains (GFP-DGK C1Am and C1Bm) and lacking the C1A or C1B domain (GFP-DGK C1A and C1B) RESULTS showed lower but significant activity than wild-type GFP-DGK. Inter- Changes in Subcellular Localization of DGK—During the cultiva- estingly, mutation in the C1A domain rather than in the C1B domain tion of CHO-K1 cells expressing GFP-DGK, we observed different affected the kinase activity. All the kinase-negative mutants, GFP- localization of GFP-DGK; some cells had GFP-DGK in the nucleus DGK KN, KNC1Am, and KNC1Bm, whose Gly-491 in the ATP bind- and some did not. Specifically, just after the transfection many of the ing site was replaced to Asp, showed no kinase activity. CHO-K1 cells express GFP-DGK only in the cytoplasm (Fig. 1A, Despite no kinase activities, both GFP-DGK KN and DGK-GFP CHO-K1, type A) but 2–3 days later, this enzyme was localized equally were localized in the nucleus as well as the cytoplasm of CHO-K1 cells, in the cytoplasm and nucleus (Fig. 1A, CHO-K1, type B). Here, we but the transportation of GFP-DGK KN into the nucleus was slower considered the possibility that the nuclear localization of GFP-DGK than wild type (Fig. 3, GFP-DGK and GFP-DGK KN). On the other was due to the degradation, because GFP itself is localized in the nucleus hand, GFP-DGK C1Am and C1Bm did not show the increase in the and the cytoplasm as shown in below in Fig. 4C. However, no degraded number of cells expressing the mutants equally in the cytoplasm and the product of GFP-DGK was found even at day 3 by immunoblotting nucleus, although they possessed significant kinase activity (Fig. 3, GFP- using GFP antibody (Fig. 1B), indicating that the enzyme is transported DGK C1Am and GFP-DGK C1Bm). Similarly, GFP-DGK C1A, from the cytoplasm to the nucleus during cultivation. Therefore, we C1B, KNC1Am, and KNC1Bm did not change their localization (Fig. investigated time-dependent changes of GFP-DGK localization. Fig. 3, GFP-DGK C1A, GFP-DGK C1B, GFP-DGK KNC1Am, and 1C shows that the number of the CHO-K1 cells expressing GFP-DGK GFP-DGK KNC1Bm). Table 1 summarizes the kinase activities of the only in the cytoplasm (Type A) decreased, while the number of the cells mutants and their transportation to the nucleus. These results show expressing the fusion protein both in the cytoplasm and the nucleus that there is no significant correlation between the kinase activity and (Type B) increased with time. Percentage of the type B CHO-K1 cells the nuclear transportation, suggesting that nuclear transportation of was 34% at day 1, but it increased to 53% at day 2. The ratio of type B to DGK does not require its kinase activity. type A cells changed from 0.826 at day 1 to 2.790 at day 2. This ratio On the other hand, as seen in the cases of GFP-DGK C1Am, C1Bm, increased further to 3.110 at day 3. We also carried out the same exper- C1A, and C1B and GFP-DGK KNC1Am and KNC1Bm, mutation iment using NIH3T3 cells. In NIH3T3 cells, the percentage of the type A in the C1A or C1B domains eliminated the nuclear transportation of cells and that of the type B cells are similar at day 1, but the type B cells DGK, suggesting that the C1 domain is important for the nuclear became the major population with time; it increased to 72% at day 2 and transportation of DGK and contains a nuclear transport signal. To 86% at day 3 (Fig. 1C, NIH3T3). Furthermore, we performed this exper- confirm the function of C1 domain as a nuclear transport signal and MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6157 Nuclear Transportation and Function of DGK FIGURE 4. Localization of the C1 domain and its fragments of DGK in CHO-K1 cells. A, schematic illustration of the C1 domain and its fragments used. C1 domain is from 269 to 380 aa, C1A domain plus Hinge is from 269 to 333 aa, Hinge plus C1B domain is from 319 to 380 aa, C1A domain is from 269 to 318 aa, Hinge is from 319 to 333 aa and C1B domain is from 334 to 380 aa of rat DGK. B, localization of FLAG-tagged C1 domain and its fragments in CHO-K1 cells. The transfected cells were cultured for 24 h and fixed. The FLAG fusion proteins were detected by mouse anti-FLAG antibody and Cy3-labeled anti-mouse IgG. C, localization of GFP-tagged C1 domain in CHO-K1 cells. Plasmids of GFP-C1 domain of DGK and GFP was transfected into CHO-K1 cells and fixed 24 h after the transfection. The fluorescence was detected by confocal microscopy. The micrographs are the images of GFP fluorescence, and the merged images represent GFP fluorescence and Nomarski images. identify which part of the C1 domain is important for the function, we localized in the nucleus but the other fragments did not show nuclear generated six fragments from the C1 domain fused with FLAG; entire localization (Fig. 4B). Similarly, GFP-tagged C1 domain was localized C1 domain, C1A domain plus Hinge, Hinge plus C1B domain, C1A specifically in the nucleus, whereas GFP alone was seen throughout the domain, Hinge and C1B domain (Fig. 4A) and investigated their local- cells (Fig. 4C). These results represent that the entire C1 domain is ization using FLAG antibody. The entire C1 domain was dominantly necessary to act as a nuclear localization signal at least in DGK. 6158 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK FIGURE 5. Differences in size of the CHO-K1 cells expressing GFP-DGK, GFP-DGK KN, GFP-DGKC1B, and GFP-DGK KNC1Am. Diameters of CHO-K1 cells expressing GFP, GFP-DGK, and mutants were measured 72 h after the transfection. The number of cells of different diameter was counted (total number of each cell type was 100). In the case of GFP, GFP-DGK, and GFP-DGK KN, the cells expressing them both in the cytoplasm and the nucleus were measured, whereas the cells expressing them only in the cytoplasm were selected in the case of GFP-DGK C1B and GFP-DGK KNC1Am. Typical images of GFP, GFP-DGK, and mutants in CHO-K1 cells are also shown to represent their localization and cell size. Furthermore, typical cross-section images and average thickness are shown on the right side. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6159 Nuclear Transportation and Function of DGK TABLE 2 The cell expressing CFP-DGK KN-NLS had a tendency to die, com- Sizes of the stable cell lines of GFP, GFP-DGK , GFP-DGK KN, and pared with control cells. Doubling time of the cells transiently express- GFP-DGK KNC1Am ing CFP-DGK KN-NLS was 17.36  1.99 h (n 25), whereas that of The cells were spread onto grass bottom dishes. After being cultured for 72 h, the control cells was 15.58  1.69 h (n 50). These results demonstrated cells were fixed, and the size of each cell was measured using LSM510 software. that nuclear GFP-DGK KN causes the disorder of cell cycle. 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 Therefore, we measured cell cycle of GFP-DGK KN by flow cytom- chosen. On the other hand, the cells expressed only in the cytoplasm were selected etry and compared it with control cell lines expressing GFP alone and in the case of GFP-DGK KNC1Am. GFP-DGK KNC1Am, the latter of which is inactive and cytosolic. At Average of cell size 2 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 GFP 333.84 GFP-DGK (localizes in cytoplasm and nucleus) 391.67 KNC1Am stable cells, indicating that nuclear, but not cytosolic DGK GFP-DGK KN (localizes in cytoplasm and nucleus) 1332.5 KN affected the cell cycle. Then, we analyzed the cell cycle more pre- GFP-DGK KNC1Am (localizes in cytoplasm) 412.67 cisely using GFP and GFP-DGK KN stable cells synchronized at the beginning of S phase by the double thymidine block method (32). Prior Physiological Functions of Nuclear DGK—To investigate physiologi- to the experiment, we determined proper concentration of thymidine cal function of DGK in the nucleus, we first measured the size of CHO-K1 for synchronizing the cells (supplemental Fig. S1). In the case of GFP- cells transiently expressing GFP, GFP-DGK, or GFP-DGK KN, because DGK KN stable cell lines, treatment with 2.5 mM thymidine resulted in we had noticed that the size of cells expressing DGK KN were bigger than partial G arrest, and 5 mM thymidine exerted a much clearer effect. For that of cells expressing wild-type DGK. Apparently, the diameter of the GFP stable cell lines, G arrest was not induced at 5 mM, and 10 mM was typical CHO-K1 cells expressing GFP and GFP-DGK was 20–30 m, needed for complete G arrest. From these results, we decided to per- whereas that of GFP-DGK KN was 30–40 m (Fig. 5, left side), suggesting form double thymidine block at 10 mM for GFP stable cells and 5 mM for the importance of DGK activity in cell shape. However, it is not clear GFP-DGK KN cells for cell-cycle analysis. whether the difference in the cell size was due to the lack of DGK the At 4 h after washing out thymidine, 37% of the control cells tran- activity in the nucleus or in the cytoplasm because GFP-DGK KN sited to G /M phase, whereas the cells expressing GFP-DGK KN in expressed both in the cytoplasm and the nucleus. We, therefore, further G /M phase were only 17% (Fig. 7). 70% of the control cells were in examined the size of cells expressing DGK mutants only in the cytoplasm to G /M phase at 5 h, but it took 6–7 h for the population of GFP-DGK examine an effect of cytoplasmic DGK activity on the cell size. We used the KN in G /M phase to reach maximum. The GFP stable cells almost cells expressing GFP-DGK KNC1Am, because the mutants has no kinase returned to G phase at 7 h, and the size of population of them in G 1 1 activity and localizes only in the cytoplasm. On the other hand, the cells phase reached 60% at 8 h, the biggest value through this experiment. On expressing GFP-DGK C1B were used as the cells with cytoplasmic the other hand, 7 h after the release, 51% of GFP-DGK KN stable kinase activity (Figs. 2C and 3). No differences were found in the size of cells cells still remained in G /M phase, and only 14% cells were in G phase. 2 1 expressing GFP-DGK KNC1Am and GFP-DGK C1B; the size of cells Finally, a major population of GFP-DGK KN transited to G phase at expressing GFP-DGK C1B and KNC1Am were similar to that of GFP- 9 h; it was delayed by 2 h compared with the GFP stable cells. These or GFP-DGK-expressing cells (Fig. 5, left side). Furthermore, we measured results clearly reveal that the S phase of GFP-DGK KN was extended, the thickness of the CHO-K1 cells transiently expressing these proteins to suggesting that the extension causes enlargement of the cell. investigate whether overexpression of DGKKN flattened the cells and/or The disorder of cell cycle by nuclear expression of DGK KN sug- resulted in increase in cell volume. As shown in Fig. 5, the cells expressing gested that alteration of the cell cycle affects the intracellular localiza- GFP-DGK KN were slightly flatter than those of the cells expressing other tion of DGK. Therefore, we observed the localization of GFP-DGK in proteins. In addition, volume of the GFP-DGK KN cells was 1.36-fold; the stable cells after serum starvation for 24 h. Under control condi- the average of the diameter and thickness of GFP-DGK KN cells were 34.7 tions, the percentage of the stable cells expressing GFP-DGK both in m and 7.98 m, whereas those of the other cells were 28.86 m and 8.46 the cytoplasm and the nucleus was 34% and that expressing only in the m, respectively. These results indicated that the overexpression of DGK cytoplasm was 51% of all cells (Fig. 8A, control). The number of the cells KN affected the cell shape and volume. To confirm this, we further made expressing GFP-DGK both in the cytoplasm and the nucleus (type B) stable CHO-K1 cell lines of GFP, GFP-DGK, GFP-DGK KN, and GFP- increased from 35% to 54% by serum starvation. In contrast, the number DGK KNC1Am and compared the size of the cells. The stable cells of the cells expressing the fusion protein only in the cytoplasm (type A) expressing GFP-DGK KN in the nucleus and the cytoplasm were remark- decreased from 51% to 34%. The ratio of type B to type A increased from ably bigger than the others (Table 2). Importantly, the size of cells express- 0.692 to 1.557. These results indicate that GFP-DGK is transported ing GFP, GFP-DGK, and GFP-DGK KNC1Am were almost same. These from the cytoplasm to the nucleus under serum-starved conditions. results indicate that dominant negative effect on the nuclear DGK, but not However, these experiments were carried out under artificial condi- cytoplasmic DGK, influences the size and volume of cells. tions using exogenously expressed GFP-DGK. Therefore, to confirm Next, to elucidate the mechanism of DGK KN-induced enlargement nuclear transportation of endogenous DGK, we performed immuno- of cells, we compared proliferation of the stable cell lines (Fig. 6). Fig. 6 staining using anti-DGK antibody. In many control cells, endogenous shows that the DGKKN cells stated the division more slowly than the DGK was localized in the cytoplasm but not in the nucleus (Fig. 8B, others and that doubling time of GFP-DGKKN stable cells was 19.1 h, control). After 24-h serum starvation, DGK localized in the nucleus as which was calculated from its regression line. On the other hand, those of GFP, GFP-DGK, and GFP-DGKKNC1Am cells were 15.4, 15.8, much as in the cytoplasm (Fig. 8, serum starvation), indicating that, as well as GFP-DGK, endogenous DGK can be transported to the and 15.8 h. These results indicated that the proliferation rate of GFP- DGK KN was slightly slower than others. To directly investigate inhib- nucleus, and the nuclear transportation is induced by serum starvation. itory effect of nuclear DGK KN on the proliferation, we constructed These results, together with the inhibitory effect of DGKKN on cell CFP-DGK KN having nuclear localization signal (CFP-DGK KN- cycle, strongly suggest an important physiological function of nuclear NLS) and CFP-NLS as control and compared their proliferation rate. DGK in the cell cycle regulation. 6160 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK FIGURE 6. Effect of nuclear expression of DGK KN on the proliferation. A, proliferation of the CHO-K1 cells stably expressing GFP, GFP-DGK, GFP-DGK KN, and GFP-DGK KNC1Am. Each stable cell line was split into dishes at 0.2  10 cells/dish. Cells were harvested after 24-, 48-, and 72-h incubations and stained by 0.4% Trypan blue in PBS(). Then the numbers of living cells were counted. Each point and vertical bar indicates the mean  S.D. of six independent experiments. The regression coefficient of GFP-DGK KN was significantly different from those of the other three (p  0.001). B, live imaging of cell division of HeLa cells expressing CFP-NLS and CFP-DGKKN-NLS. The cell division of HeLa cells transiently expressing CFP- DGK KN NLS and CFP-NLS were observed as described under “Experimental Procedures.” Typical images of CFP-DGKKN-NLS and CFP-NLS cells are shown. Numbers on the right side show average of doubling time (n 25 for CFP-DGKKN-NLS, n 50 for CFP-NLS), which is from the first division to the second one. *, difference between the average of doubling time was significant (p  0.001). MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6161 Nuclear Transportation and Function of DGK FIGURE 7. Cell-cycle analysis of the stable cell lines of GFP, GFP-DGK KN, and GFP-DGKKN C1Am by flow cytometry. A, cell-cycle analysis of the stable cell lines of GFP, GFP-DGK KN, and GFP-DGKKN C1Am. 0.2  10 cells of each stable cell lines were spread on 10-cm dishes. The cells were synchronized by serum starvation for 24 h. After 24 h of serum restoration, the cells were treated with trypsin-EDTA and collected in a 1.5-ml tube. The collected cells were fixed by 70% EtOH and incubated with RNase A, and DNA was stained by propidium iodide. The cell cycle was analyzed by a FACSCalibur. B, cell-cycle analysis of the stable cell lines of GFP and GFP-DGK KN released from the double thymidine block. 0.2 10 cells of each stable cell line of GFP and GFP-DGKKN were spread on 10-cm dishes. The cells were synchronized at the beginning of the S phase with 10 (for GFP stable cells) or 5 mM (for GFP-DGK KN stable cells) thymidine. Every hour after the release, the cells were treated with trypsin-EDTA and collected in a 1.5-ml tube, and then subjected to flow cytometric analysis. DISCUSSION 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- In this report, we showed for the first time that DGK is transported DGK KNC1Bm versus GFP-DGK C1Bm in Fig. 3). These results suggest from the cytoplasm to the nucleus (Fig. 1). The nuclear transportation of that the kinase activity, although not essential, may have some roles in the DGK was independent of kinase activity of DGK (Fig. 3 and Table 1), but nuclear transportation of DGK. the mutant lacking kinase activity (DGK KN) showed much slower nuclear transportation than that of wild type. The mutation eliminating Instead of kinase activity, the C1 domain of DGK was essential for the kinase activity of GFP-DGK C1Am or GFP-DGK C1Bm made a remark- nuclear transportation of DGK. Namely, none of the C1 domain mutants 6162 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK (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 local- ized 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 flu- orescence 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 FIGURE 8. Effect of serum starvation on the localization of exogenous GFP-DGK inhibit the endogenous nuclear DGK by dominant-negative effects. and endogenous DGK in CHO-K1 cells. A, the change in subcellular localization of The cells expressing GFP-DGK KN were larger in the size (Fig. 5 and 5 GFP-DGK in the GFP-DGK stable cell line by serum starvation. 3.2  10 cells were spread onto glass bottom dishes and cultured overnight. The cells were further cultured Table 2), proliferated slowly (Fig. 6), and their S phase was extended for an additional 24 h in the presence (control) and absence of serum (free 24 h) and fixed. (Fig. 7), suggesting that enlargement of cell size is induced by protein Moreover, 100 cells were observed, and the number of the cells expressing GFP-DGK synthesis during extended S phase. In addition, serum starvation only in the cytoplasm (shaded bars), abundantly in the cytoplasm (), and equally in the cytoplasm and the nucleus (f) were counted. Same experiment was independently induced nuclear transportation of endogenous DGK and GFP-DGK performed three times and the averages  S.E. are plotted as percentage. Numbers stably expressing in the CHO-K1 cells (Fig. 8). These results strongly 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 implicate that DGK in the nucleus is involved in regulation of cell intact cells under serum starvation conditions. CHO-K1 cells were spread onto glass cycle. bottom dishes and cultured overnight. After being cultured for an additional 24 h in the Involvement of DGK in cell-cycle regulation is supported by several 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- reports as follows. First, inhibition of DGK activity prevents transition DGK antibody. from G to S phase (39). Second, the involvement of other DGK sub- types such as DGK in the cell cycle has also been reported, although confirmed the expression of DGK in various tissues, including kidney, there are some differences; COS-7 cells expressing DGK showed the muscle, and other tissues, in addition to brain (supplemental Fig. S2). increase in the size of G cell population and decrease in that of G /M Therefore, at least in these cells and tissues, it is expected that nuclear 1 2 cell population but no effects of DGK KN on the cell cycle were DGK is involved in cell-cycle regulation. In fact, regulation of differ- detected (21). The differences may be due to subtype specificity and/or entiation by DGK in HL-60 cells has been suggested (41, 43). different cell types used. However, it still remains to be solved how DGK can regulate the cell Both DGK and DGK are abundantly expressed in the brain (40), cycle. The amount of nuclear phosphatidylinositols, including phos- although it is unclear about their functions as a regulator of the cell cycle phatidylinositol 4,5-bisphosphate, fluctuates during the cell cycle (22), in the central nervous system. However, expression of DGK has been and the DG mass in the nucleus increases in G /M phase (27). Further- recently reported in HL-60, U937, and NIH3T3 cells (41–43). We also more, IIPKC translocates into the nucleus and phosphorylates lamin B MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6163 Nuclear Transportation and Function of DGK (1996) J. Biol. Chem. 271, 10230–10236 in the G /M phase (44). These reports suggest that the DG derived from 18. Ding, L., Traer, E., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1998) phosphatidylinositol 4,5-bisphosphate recruits PKC to regulate G /M J. Biol. Chem. 273, 32746–32752 phase. In other words, regulation of nuclear DG mass is important for 19. Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, cell-cycle regulation, and DGK might be involved in the control of M., Takenawa, T., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422–10428 20. Bregoli, L., Baldassare, J. J., and Raben, D. M. (2001) J. Biol. Chem. 276, 23288–23295 nuclear DG. Alternatively, PA produced by DGK may have some func- 21. Topham, M. K., Bunting, M., Zimmerman, G. A., McIntyre, T. M., Blackshear, P. J., tion in cell-cycle regulation. In fact, the amount of PA in the nucleus also and Prescott, S. M. (1998) Nature 394, 697–700 changes by the stimulation of -thrombin, which has a mitogenic effect 22. York, J. D., and Majerus, P. W. (1994) J. Biol. Chem. 269, 7847–7850 (45). Accordingly, the mechanism of cell-cycle regulation by DGK is an 23. D’Santos, C. S., Clarke, J. H., and Divecha, N. (1998) Biochim. Biophys. Acta 1436, 201–232 important issue to be solved next. 24. Divecha, N., Banfic, H., and Irvine, R. F. (1991) EMBO J. 10, 3207–3214 In conclusion, the present results indicate that DGK is transported 25. Divecha, N., Treagus, J., Vann, L., and D’Santos, C. (1997) Semin. Cell Dev. Biol. 8, from the cytoplasm to the nucleus via its C1 domain and suggest that 323–331 DGK is involved in cell-cycle regulation. 26. Banfic, H., Zizak, M., Divecha, N., and Irvine, R. F. (1993) Biochem. J. 290, 633–636 27. Deacon, E. M., Pettitt, T. R., Webb, P., Cross, T., Chahal, H., Wakelam, M. J., and Lord, J. M. (2002) J. Cell Sci. 115, 983–989 Acknowledgments—We thank Takumi Kawasaki (Biosignal Research Center, 28. Shirai, Y., Segawa, S., Kuriyama, M., Goto, K., Sakai, N., and Saito, N. (2000) J. Biol. Kobe University) for assistance with RT-PCR and Dr. Hideyuki Saya (Depart- Chem. 275, 24760–24766 ment of Tumor Genetics and Biology, Graduate School of Medical Sciences, 29. Shindo, M., Irie, K., Masuda, A., Ohigashi, H., Shirai, Y., Miyasaka, K., and Saito, N. 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Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus *

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10.1074/jbc.m509873200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp. 6152–6164, March 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Nuclear Transportation of Diacylglycerol Kinase  and Its □ S Possible Function in the Nucleus Received for publication, September 7, 2005, and in revised form, December 27, 2005 Published, JBC Papers in Press, December 30, 2005, DOI 10.1074/jbc.M509873200 ‡ ‡1 ‡ ‡ ‡ Takehiro Matsubara , Yasuhito Shirai , Kei Miyasaka , Takuya Murakami , Yasuto Yamaguchi , ‡ § § § ¶ ¶ Takehiko Ueyama , Masahiro Kai , Fumio Sakane , Hideo Kanoh , Toshiaki Hashimoto , Shinji Kamada , ¶ ‡2 Ushio Kikkawa , and Naoaki Saito ‡ ¶ From the Laboratory of Molecular Pharmacology and Laboratory of Biochemistry, Biosignal Research Center, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501 and the Department of Biochemistry, Sapporo Medical University School of Medicine, West-17, South-1, Chuo-Ku, Sapporo 060-8556, Japan Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to consists of at least nine subtypes (2). Although all DGKs have cysteine- phosphatidic acid, and both lipids are known to play important rich repeats similar to the C1A and C1B domains of PKCs in the N roles in lipid signal transduction. Thereby, DGKs are considered to terminus and a catalytic domain in the C terminus, they are divided into be a one of the key players in lipid signaling, but its physiological five groups on the primary structure of these DGKs. Type I DGKs, function remains to be solved. In an effort to investigate one of nine including DGK,-, and -, have EF-hand motifs and two cysteine-rich subtypes, we found that DGK came to be localized in the nucleus regions (C1 domain) in the regulatory domain (12, 13), whereas Type II with time in all cell lines tested while seen only in the cytoplasm at DGKs, DGK and -, have a pleckstrin homology domain instead of the the early stage of culture, indicating that DGK is transported from EF-hand motif in addition to the C1 domain (14, 15). The separated the cytoplasm to the nucleus. The nuclear transportation of DGK catalytic domains of DGK and - are characteristic of Type II DGK. didn’t necessarily need DGK activity, but its C1 domain was indis- Type III, DGK, has only one C1 domain in the regulatory domain (16). pensable, suggesting that the C1 domain of DGK acts as a nuclear Myristoylated alanine-rich protein kinase C substrate phosphorylation transport signal. Furthermore, to address the function of DGK in site-like region and four ankyrin repeats are unique domains in Type IV the nucleus, we produced stable cell lines of wild-type DGK and DGK (17, 18). The final group, type V, includes DGK, which has three mutants, including kinase negative, and investigated their cell size, cysteine-rich regions and a pleckstrin homology domain with overlap- growth rate, and cell cycle. The cells expressing the kinase-negative ping Ras-associating domain (19). The DGKs are thought to be involved mutant of DGK were larger in size and showed slower growth rate, in development, differentiation, construction of neural network and and the S phase of the cells was extended. These findings implicate immunity, etc. However, subtype specific function and regulation that nuclear DGK regulates cell cycle. mechanisms of DGKs are not clear. Several groups have reported many different localization and trans- location of DGKs, possibly contributing to their subtype-specific func- Diacylglycerol (DG) is a second messenger regulating various cellu- tions. In an effort to elucidate the function of DGK, we unexpectedly lar responses (1, 2). One of the important roles of DG is an activating of found that GFP-fused DGK (GFP-DGK) became localized in the protein kinase C (PKC) (1, 3, 4). DG is physiologically produced as a result of the signal-induced hydrolysis of phosphatidylinositol by phos- nucleus as well as the cytoplasm a few days after transfection but was pholipase C. The generated DG is phosphorylated to phosphatidic acid localized mainly in the cytoplasm just after expressed in CHO-K1 cells. by diacylglycerol kinase (DGK) or metabolized by DG lipase (2, 5, 6). Although nuclear transportation of DGK has never been reported, Thus, DGK is an important enzyme to inactivate PKC by reducing the expression of DGK and DGK in the nucleus has been already DG level, contributing to regulating of the cellular response. In addition, described (20, 21). In addition, DGK is thought to be involved in the phosphatidic acid itself activates PKC (7), phosphatidylinositol regulation of cell cycle (21). These findings, together with the facts that 4-phosphate 5-kinase (8, 9), and mammalian target of rapamycin (10), phosphatidylinositol turnover exists within the nucleus and DG may be and modulates Ras GTPase-activating protein (11). involved in the regulation of cell cycle (22–27), suggest that DGK has Molecular cloning studies revealed that mammalian DGK family some physiological function in the nucleus. However, mechanism of the nuclear transportation and physiological functions of DGK are *This work was supported by grants from the 21st Century Center of Excellence Program unknown. We, therefore, investigated molecular mechanism and phys- of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from iological significance of nuclear transportation of DGK. 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 EXPERIMENTAL PROCEDURES the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S Materials—CHO-K1 cells were donated from Dr. M. Nishijima The on-line version of this article (available at http://www.jbc.org) contains supple- mental Figs. S1 and S2. (National Institute of Health, Tokyo, Japan). COS-7 cells, SH-SY5Y To whom correspondence may be addressed. Tel.: 81-78-803-5961; Fax: 81-78-803- cells, and HeLa cells were purchased from the RIKEN Cell Bank. Fetal 5971; E-mail: [email protected]. To whom correspondence may be addressed. Tel.: 81-78-803-5961; Fax: 81-78-803- bovine serum, RNase A, and anti-FLAG M2 monoclonal antibody were 5971; E-mail: [email protected]. obtained from Sigma. FuGENE 6 Transfection reagent was obtained The abbreviations used are: DG, diacylglycerol; PKC, protein kinase C; DGK, diacylglyc- erol kinase; CHO, Chinese hamster overly; C1, conserved region 1; C1A, first half of C1 from Roche Applied Science. Propidium iodide was obtained from domain; C1B, second half of C1 domain; KN, kinase negative; COS, African green Wako (Osaka, Japan). FluoroLink Cy3-labeled goat anti-mouse IgG was monkey kidney; GFP, green fluorescent protein; NLS, nuclear localization signal; PBS, phosphate-buffered saline; aa, amino acid(s). purchased from Amersham Biosciences. We produced anti-GFP anti- 6152 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 This is an Open Access article under the CC BY license. Nuclear Transportation and Function of DGK 4 32 body ourselves. [- P]ATP was obtained from ICN Biomedicals CGAATTCATGGTGAAAACATACTCC-3 and 5-TGAGATCTG- (Irvine, CA). A TLC plate was obtained from Merck (Darmstadt, Ger- CACGCTTGCACAAT-3. The primers for C1A domain (805–954 bp) many). Geneticin was purchased from Invitrogen. were 5-TTGAATTCATGCCACGCCTGGACCCCTGA-3 and 5-T- Cell Culture—CHO-K1 cells were cultured in Ham’s F-12 medium TAGATCTCTGCATCACCTCACC-3. The primers for Hinge (Nacalaitesque, Japan). COS-7 and NIH3T3 cells were cultured in Dul- (955–990 bp) were 5-CCGAATTCATGGTGAAAACATACTCC-3 becco’s modified Eagles’ medium (Nacalaitesque, Japan). SH-SY5Y cells and 5-TTAGATCTCTGCATCACCTCACC-3. The primers for C1B were cultured in Dulbecco’s modified Eagles’ medium/Ham’s F-12 domain (1000–1140 bp) were 5-TTGAATTCATGCACGCGTGGG- medium (1:1) (Invitrogen). All cells were cultured at 37 °C in humidified TGGAA-3 and 5-TGAGATCTGCACGCTTGTCGACATA-3. atmosphere containing 5% CO . All media contained 25 mM glucose, Transfection—Cells (2.0 10 cells/dish) on glass-bottom dish (Mat- TM and all were buffered with 44 mM NaHCO and supplemented with 10% Tek Corp., Ashland, MA) were transfected using 3 l of FuGENE 6 fetal bovine serum (Sigma), penicillin (100 units/ml), and streptomycin transfection reagent (Roche Applied Science) and 1 g of DNA accord- (100 g/ml) (Invitrogen). The fetal bovine serum used was not heat- ing to the manufacturer’s protocol. Transfected cells were cultured at inactivated. The media for SH-SY5Y cells was added with 1% GlutaMA- 37 °C for 24 h before use. TM X -I Supplement (Invitrogen). Immunostaining—CHO-K1 cells transfected cDNA coding FLAG Constructs of Plasmids Encoding DGK-fused GFP and Mutants— fusion proteins as described above, and those without transfection were The constructs encoding DGK having GFP at its N terminus (GFP- fixed for1hat room temp with 4% paraformaldehyde, 0.2% picric acid in DGK) or at C terminus (DGK-GFP) and mutants having substitution 0.1 M phosphate buffer and permeabilized with 0.3% TritonX-100 in of Cys-285 to Gly in the C1A domain (GFP-DGK C1Am) or Cys-348 to 0.01 M PBS for 15 min at room temp. Cells were sequentially incubated Gly in the C1B domain (GFP-DGK C1Bm) were previously described with 10% normal goat serum, mouse anti-FLAG antibody (Sigma), or (28). The constructs encoding the mutants lacking the C1A or C1B anti-DGK antibody, and then Fluorolink Cy3-labeled goat anti-mouse domain (GFP-DGK C1A or GFP-DGK C1B) were also described antibody. At each step, transfected cells were washed three times with previously (29). Kinase negative mutant of DGK (GFP-DGK KN) was 0.01 M PBS containing 0.03% Triton X-100 (PBS-T) for 5 min. produced by substitution of Gly-491 to Asp. Briefly, using the plasmid For preparation of anti-DGK antibody, an oligopeptide correspond- encoding GFP-DGK, site-directed mutagenesis was performed ing to amino acids 778–791 of human DGK (31) was used for antigen. according to the manufacturer’s recommended protocol with an ExSite This antibody was purified by antigen-immobilized affinity column. We PCR-based site-directed mutagenesis kit (Stratagene). The primers finally confirmed that the purified DGK antibody has no cross-reac- were 5-GCTGGATCCTGGATTGCTTTGACAAGG-3 and 5-CA- tions with pig DGK or rat DGK. ACTGTGTCATCTCCGCCACAGGCAA-3. The mutation was con- Confocal Microscopy—The fluorescence of Cy3 and GFP were firmed by verifying its sequence. Furthermore, to make kinase-negative observed under confocal laser scanning fluorescent microscopy (Carl mutants having substitution of Cys-285 or Cys-348 to Gly (GFP-DGK Zeiss, Jena, Germany). The GFP fluorescence was monitored at 488 nm KNC1Am and GGFP-DGK KNC1Bm), the cDNA of DGK KN was argon laser excitation with 515 nm long pass barrier filter. Cy3 fluores- digested with SalI and SmaI and then subcloned into the SalI and SmaI cence was monitored at 543 nm HeNe 1 excitation with a 560–615 nm site in the plasmid encoding GFP-DGK C1Am and GFP-DGK C1Bm, band pass filter. respectively. Upon quantitating, the subcellular distribution of the proteins fused In addition, to construct DGK KN having a nuclear localization GFP, which we described below. The cells in which the ratio of GFP fluo- signal (NLS), cDNA fragments of DGK KN with BspEI site at the N rescence intensity of cytoplasm and nucleus was under 0.3 were defined as terminus and XhoI site at the C terminus were produced by a PCR using “only in the cytoplasm,” and the cells with the ratio of 0.3–0.8 were defined cDNA for kinase-negative mutant of DGK constructed as described as “abundantly in the cytoplasm.” In the case the ratio was over 0.8, the cells above. The PCR products were first subcloned into a pGEM T-Easy were defined as “equally in the cytoplasm and the nucleus.” vector (Promega, Madison, WI). After digestion with BspEI and XhoI, Immunoblotting and Kinase Assay—Plasmids (32 g) were electro- the cDNA encoding DGK KN was subcloned into the BspEI and XhoI porated into COS-7 cells using a GenePulser (Bio-Rad, 975 microfarads, site in the pECFP-Nuc vector (Clontech). The primers were 5-AATC- TM 220 mV) or transfected into NIH3T3 cells using FuGENE 6 transfection CGGGATGAGTGACGGGCAATGG-3 and 5-GCTCGAGCGT- reagent. After being cultured, the cells were harvested and centrifuged at CCTTGAACGGCTTTTCCTC-3. 5500  g for 3 min. The cells were resuspended in homogenizing buffer Constructs of the Plasmids Encoding C1 Domain Fragments of (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 50 mM Tris-HCl, 200 g/ml DGK—cDNA fragments of C1 domain with EcoRI site in the N termi- leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% TritonX-100, pH 7.4) nus and BamHI site in the C terminus were produced by a PCR using and sonicated (UD-210 Tomy, Japan, output 3, 15 s, 2 times). cDNA for rat DGK as the template (30). The PCR products were first For immunoblotting, the samples were subjected to 7.5% SDS- subcloned into a pGEM T-Easy vector (Promega). After digestion with polyacrylamide gel electrophoresis, followed by blotting onto a poly- EcoRI and BamHI, the cDNA-encoding C1 domain was subcloned into vinylidene difluoride membrane (Millipore, Bedford, MA). Nonspe- TM the EcoRI and BglII sites in the p3xFLAG-CMV -14 expression vector cific binding sites were blocked by incubation with 5% skim milk in (Sigma) or pEGFPC2 (Clontech). The primers for C1 domain (805– PBS-T at 4 °C overnight. The membrane was incubated with anti- 1140 bp) were 5-TTGAATTCATGCACGCCTGGACCCGA-3 and GFP antibody for 1 h at room temperature. After washing with 5-TGAGATCTGCACGCTGTCGACCAAT-3. The primers for C1A PBS-T, the membrane was incubated with peroxidase-labeled anti- domain plus Hinge (805–999 bp) were 5-TTGAATTCATGCACGG- rabbit IgG (Jackson, ImmunoResearch Laboratories, West Grove, CCTGGACCCTG-3 and 5-TTAGATCTCTGCATCACCTCACC- PA) for 30 min. After three rinses with PBS-T, the immunoreactive 3. The primers for Hinge plus C1B domain (955–1140 bp) were 5-C- bands were visualized using a chemiluminescence detection kit (ECL, Amersham Biosciences). To determine the kinase activity of DGK and mutants, appropriate Y. Yamaguchi, Y. Shirai, T. Matsubara, N. Ohshiro, K. Yoshino, K. Yonezawa, Y. Ono, and N. Saito, manuscript in preparation. volumes of the homogenate samples, which contain comparative MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6153 Nuclear Transportation and Function of DGK FIGURE 1. Changes in subcellular localization of GFP-DGK in CHO-K1, NIH3T3, and SH-SY5Y cells. A, different type of DGK localization in CHO-K1 cells, NIH3T3 cells, and SH-SY5Y cells. Plas- mids of GFP-DGK were transfected by lipofection using FuGENE in respective cell lines. 24 h after transfection, some cells express GFP-DGK only in cytoplasm (Type A), and the fusion proteins are localized equally in the cytoplasm and the nucleus (Type B). B, immunoblot analysis of GFP-DGK at days 1 and 3 after transfection by anti-GFP anti- body. The plasmids were transfected to NIH3T3 cells by lipofection. The cells were harvested 1 or 3 days after transfection. The lysates of the trans- fected cells were subjected to SDS-PAGE and Western blotting as described under “Experimen- tal Procedures.” The immunoreactive bands were detected by using anti-GFP antibody. The molec- ular mass of marker protein is indicated on the left. C, time-dependent change of DGK localization in CHO-K1, NIH3T3, and SH-SY5Y cells. More than 100 cells were observed at 24 h (day 1), 48 h (day 2), and 72 h (day 3) after transfection, respectively, and the number of the cells expressing GFP-DGK only in the cytoplasm (shaded bars), abundantly in the cytoplasm () and equally in the cytoplasm and the nucleus (f) were counted. Same experi- ment was independently performed three times, and the averages S.E. are plotted as percentage. Numbers below the graph show the ratio of the cells expressing GFP-DGK in both in the cyto- plasm and the nucleus (Type B) to only in the cyto- plasm (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 day1(p  0.05). amounts of the fusion protein of DGK or mutants assessed by immu- Measurement of the Diameter and Thickness of the Cells Transiently noblotting, were subjected to octyl glucoside mixed-micelle assay (14) Expressing Fluorescent Proteins—Plasmids (5.5 g) encoding GFP- by subtle modification. 1-Steroyl-2-arachidonoyl-sn-glycerol (Biomol, DGK, GFP-DGK KN, GFP-DGK C1B, GFP-DGK KNC1Am, and Plymouth Meeting, PA) was used as substrate. The radioactivity of GFP alone were transfected into CHO-K1 cells by lipofection as described phosphatidic acid was separated on 20-cm Silica gel 60 TLC plates above. Diameters of the respective cells were measured by LSM510 soft (Merck) using a chloroform:methanol:acetic acid (65:15:5) solution and ware under confocal microscopy. The thickness of the cells was measured detected by using a BAS2500 (Fujix, Tokyo, Japan) using re-constructed image of three-dimensional sections. 6154 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK 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. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6155 Nuclear Transportation and Function of DGK FIGURE 3. Changes in subcellular localization of DGK mutants in CHO-K1 cells. The number of the cells expressing GFP-DGK KN, DGK-GFP, GFP-DGK C1Am, GFP-DGK C1Bm, GFP-DGK C1A, GFP-DGK C1B, GFP-DGK KNC1Am, and GFP-DGK KNC1Bm only in the cytoplasm (shaded bars), abundantly in the cytoplasm () and equally in the cytoplasm and the nucleus (f), were counted at each time point and shown as percentage. Numbers below the graph show the ratio of numbers of the cells expressing each mutants or DGK-GFP both in the cytoplasm and the nucleus to only in the cytoplasm. The insets in these graphs show the change in the ratio of the cells expressing each mutants or DGK-GFP 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  0.05). Production of Stable Cell Lines—Plasmids (5.5 g) encoding GFP- according to the manufacturer’s protocol. Geneticin (0.5 mg/ml) was DGK, GFP-DGK KN, GFP-DGK KNC1Am, and GFP alone were added to the medium 24 h after the transfection. After being cultured TM transfected by lipofection using FuGENE 6 transfection reagent for more than 24 h, the transfected cells were transferred to 96-well 6156 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK plates at 0.5 cell/well for cloning. The positive clone was identified by TABLE 1 the fluorescence under confocal microscopy. The activity of GFP-DGK , DGK -GFP, and mutants and their nuclear localization in CHO-K1 cells Proliferation of Stable Cell Lines—0.6  10 cells of each stable cell 6 The number of “” symbols in the box of the activity of DGK represents the strength lines were split on three 10-cm dishes (0.2  10 each). After 24 (day 1), of kinase activity. In the box of the localization in the nucleus, “” represents the 48 (day 2), and 72 h (day 3), cells were treated with trypsin-EDTA after exogenous proteins that are localized in the nucleus, and “” represents exogenous proteins that are not in the nucleus. washed PBS() and collected by centrifugation (1250  g for 10 min, at The activity of Localization in the 4 °C). The harvested cells were resuspended in 1 ml of PBS(), stained DGK nucleus with 0.4% Trypan blue in PBS(), and counted. The doubling time of GFP-DGK the lines were calculated from simple regression based on the number of GFP-DGK KN the cells. Difference of correlation coefficients of the regression lines DGK-GFP GFP-DGK C1Am was determined by testing the t value. GFP-DGK C1Bm Live Imaging of Cell Division of HeLa Cells Expressing CFP-NLS and GFP-DGK C1A GFP-DGK C1B CFP-DGKKN-NLS—Plasmids encoding CFP-NLS and CFP-DGK GFP-DGK KNC1Am KN-NLS were transfected into HeLa cells. Images of both phase con- GFP-DGK KNC1Bm trast and CFP fluorescence were taken every 10 min from 24 to 72 h after transfection using fluorescent microscopy, BZ-8000 (Keyence, Osaka, iment using the neuroblastoma cell line, SH-SY5Y, because DGK are Japan) equipped with cultivation system, INU-KI-F1 (Tokai Hit, Shi- abundantly expressed in the brain. In SH-SY5Y cells, DGK also zuoka, Japan). Based on the images, the doubling time of respective cells, showed different types of the localization (Fig. 1A, SH-SY5Y, type A and the time period between the first and the second division, was type B). Percentage of the type B SH-SY5Y cells was 47% at day 1, and it measured. was 52.2% at day 2 with the change in ratio from 1.395 to 2.373 (Fig. 1C, Flow Cytometry—0.2  10 cells of each stable cell lines were spread SH-SY5Y). Finally, the ratio reached 4.183 at day 3. These results indi- on 10-cm dishes. The cells were synchronized at the beginning of S cate that DGK is transported to the nucleus depending on cultivation phase by double thymidine block and release protocol. Briefly, the cells periods in all cell lines tested. were treated with 10 mM (for GFP stable cells) or 5 mM (for GFP-DGK Mechanism for Nuclear Transportation of DGK—To analyze KN stable cells) thymidine for the first 18 h, followed by an interval of whether enzymatic activity is required for the nuclear transportation of thymidine-free incubation for 10 h, and the second thymidine incuba- DGK, several mutants were generated as shown in Fig. 2A, and corre- tion for 8 h (32). To release the cell cycle, the cells were washed well and lation between their activities and nuclear transportation was investi- cultured in normal medium containing serum. Every hour after the gated. Immunoblotting using anti-GFP antibody revealed that each release, cells were treated with trypsin-EDTA and collected in a 1.5-ml mutant had appropriate molecular size (Fig. 2B) and that no significant tube. Washed by PBS(), the cells were fixed by 70% ethanol for1hon degraded products were detected. Fig. 2C shows that GFP-DGK had ice, and treated sequentially by 0.25 mg/ml RNase A and 0.05 mg/ml kinase activity, whereas DGK fused GFP at the N terminus (DGK- propidium iodide. Cell cycle analysis was performed by using a FACS- GFP) had no activity as previously reported (28). The mutants having Calibur (BD Biosciences). mutations in the C1A or C1B domains (GFP-DGK C1Am and C1Bm) and lacking the C1A or C1B domain (GFP-DGK C1A and C1B) RESULTS showed lower but significant activity than wild-type GFP-DGK. Inter- Changes in Subcellular Localization of DGK—During the cultiva- estingly, mutation in the C1A domain rather than in the C1B domain tion of CHO-K1 cells expressing GFP-DGK, we observed different affected the kinase activity. All the kinase-negative mutants, GFP- localization of GFP-DGK; some cells had GFP-DGK in the nucleus DGK KN, KNC1Am, and KNC1Bm, whose Gly-491 in the ATP bind- and some did not. Specifically, just after the transfection many of the ing site was replaced to Asp, showed no kinase activity. CHO-K1 cells express GFP-DGK only in the cytoplasm (Fig. 1A, Despite no kinase activities, both GFP-DGK KN and DGK-GFP CHO-K1, type A) but 2–3 days later, this enzyme was localized equally were localized in the nucleus as well as the cytoplasm of CHO-K1 cells, in the cytoplasm and nucleus (Fig. 1A, CHO-K1, type B). Here, we but the transportation of GFP-DGK KN into the nucleus was slower considered the possibility that the nuclear localization of GFP-DGK than wild type (Fig. 3, GFP-DGK and GFP-DGK KN). On the other was due to the degradation, because GFP itself is localized in the nucleus hand, GFP-DGK C1Am and C1Bm did not show the increase in the and the cytoplasm as shown in below in Fig. 4C. However, no degraded number of cells expressing the mutants equally in the cytoplasm and the product of GFP-DGK was found even at day 3 by immunoblotting nucleus, although they possessed significant kinase activity (Fig. 3, GFP- using GFP antibody (Fig. 1B), indicating that the enzyme is transported DGK C1Am and GFP-DGK C1Bm). Similarly, GFP-DGK C1A, from the cytoplasm to the nucleus during cultivation. Therefore, we C1B, KNC1Am, and KNC1Bm did not change their localization (Fig. investigated time-dependent changes of GFP-DGK localization. Fig. 3, GFP-DGK C1A, GFP-DGK C1B, GFP-DGK KNC1Am, and 1C shows that the number of the CHO-K1 cells expressing GFP-DGK GFP-DGK KNC1Bm). Table 1 summarizes the kinase activities of the only in the cytoplasm (Type A) decreased, while the number of the cells mutants and their transportation to the nucleus. These results show expressing the fusion protein both in the cytoplasm and the nucleus that there is no significant correlation between the kinase activity and (Type B) increased with time. Percentage of the type B CHO-K1 cells the nuclear transportation, suggesting that nuclear transportation of was 34% at day 1, but it increased to 53% at day 2. The ratio of type B to DGK does not require its kinase activity. type A cells changed from 0.826 at day 1 to 2.790 at day 2. This ratio On the other hand, as seen in the cases of GFP-DGK C1Am, C1Bm, increased further to 3.110 at day 3. We also carried out the same exper- C1A, and C1B and GFP-DGK KNC1Am and KNC1Bm, mutation iment using NIH3T3 cells. In NIH3T3 cells, the percentage of the type A in the C1A or C1B domains eliminated the nuclear transportation of cells and that of the type B cells are similar at day 1, but the type B cells DGK, suggesting that the C1 domain is important for the nuclear became the major population with time; it increased to 72% at day 2 and transportation of DGK and contains a nuclear transport signal. To 86% at day 3 (Fig. 1C, NIH3T3). Furthermore, we performed this exper- confirm the function of C1 domain as a nuclear transport signal and MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6157 Nuclear Transportation and Function of DGK FIGURE 4. Localization of the C1 domain and its fragments of DGK in CHO-K1 cells. A, schematic illustration of the C1 domain and its fragments used. C1 domain is from 269 to 380 aa, C1A domain plus Hinge is from 269 to 333 aa, Hinge plus C1B domain is from 319 to 380 aa, C1A domain is from 269 to 318 aa, Hinge is from 319 to 333 aa and C1B domain is from 334 to 380 aa of rat DGK. B, localization of FLAG-tagged C1 domain and its fragments in CHO-K1 cells. The transfected cells were cultured for 24 h and fixed. The FLAG fusion proteins were detected by mouse anti-FLAG antibody and Cy3-labeled anti-mouse IgG. C, localization of GFP-tagged C1 domain in CHO-K1 cells. Plasmids of GFP-C1 domain of DGK and GFP was transfected into CHO-K1 cells and fixed 24 h after the transfection. The fluorescence was detected by confocal microscopy. The micrographs are the images of GFP fluorescence, and the merged images represent GFP fluorescence and Nomarski images. identify which part of the C1 domain is important for the function, we localized in the nucleus but the other fragments did not show nuclear generated six fragments from the C1 domain fused with FLAG; entire localization (Fig. 4B). Similarly, GFP-tagged C1 domain was localized C1 domain, C1A domain plus Hinge, Hinge plus C1B domain, C1A specifically in the nucleus, whereas GFP alone was seen throughout the domain, Hinge and C1B domain (Fig. 4A) and investigated their local- cells (Fig. 4C). These results represent that the entire C1 domain is ization using FLAG antibody. The entire C1 domain was dominantly necessary to act as a nuclear localization signal at least in DGK. 6158 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK FIGURE 5. Differences in size of the CHO-K1 cells expressing GFP-DGK, GFP-DGK KN, GFP-DGKC1B, and GFP-DGK KNC1Am. Diameters of CHO-K1 cells expressing GFP, GFP-DGK, and mutants were measured 72 h after the transfection. The number of cells of different diameter was counted (total number of each cell type was 100). In the case of GFP, GFP-DGK, and GFP-DGK KN, the cells expressing them both in the cytoplasm and the nucleus were measured, whereas the cells expressing them only in the cytoplasm were selected in the case of GFP-DGK C1B and GFP-DGK KNC1Am. Typical images of GFP, GFP-DGK, and mutants in CHO-K1 cells are also shown to represent their localization and cell size. Furthermore, typical cross-section images and average thickness are shown on the right side. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6159 Nuclear Transportation and Function of DGK TABLE 2 The cell expressing CFP-DGK KN-NLS had a tendency to die, com- Sizes of the stable cell lines of GFP, GFP-DGK , GFP-DGK KN, and pared with control cells. Doubling time of the cells transiently express- GFP-DGK KNC1Am ing CFP-DGK KN-NLS was 17.36  1.99 h (n 25), whereas that of The cells were spread onto grass bottom dishes. After being cultured for 72 h, the control cells was 15.58  1.69 h (n 50). These results demonstrated cells were fixed, and the size of each cell was measured using LSM510 software. that nuclear GFP-DGK KN causes the disorder of cell cycle. 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 Therefore, we measured cell cycle of GFP-DGK KN by flow cytom- chosen. On the other hand, the cells expressed only in the cytoplasm were selected etry and compared it with control cell lines expressing GFP alone and in the case of GFP-DGK KNC1Am. GFP-DGK KNC1Am, the latter of which is inactive and cytosolic. At Average of cell size 2 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 GFP 333.84 GFP-DGK (localizes in cytoplasm and nucleus) 391.67 KNC1Am stable cells, indicating that nuclear, but not cytosolic DGK GFP-DGK KN (localizes in cytoplasm and nucleus) 1332.5 KN affected the cell cycle. Then, we analyzed the cell cycle more pre- GFP-DGK KNC1Am (localizes in cytoplasm) 412.67 cisely using GFP and GFP-DGK KN stable cells synchronized at the beginning of S phase by the double thymidine block method (32). Prior Physiological Functions of Nuclear DGK—To investigate physiologi- to the experiment, we determined proper concentration of thymidine cal function of DGK in the nucleus, we first measured the size of CHO-K1 for synchronizing the cells (supplemental Fig. S1). In the case of GFP- cells transiently expressing GFP, GFP-DGK, or GFP-DGK KN, because DGK KN stable cell lines, treatment with 2.5 mM thymidine resulted in we had noticed that the size of cells expressing DGK KN were bigger than partial G arrest, and 5 mM thymidine exerted a much clearer effect. For that of cells expressing wild-type DGK. Apparently, the diameter of the GFP stable cell lines, G arrest was not induced at 5 mM, and 10 mM was typical CHO-K1 cells expressing GFP and GFP-DGK was 20–30 m, needed for complete G arrest. From these results, we decided to per- whereas that of GFP-DGK KN was 30–40 m (Fig. 5, left side), suggesting form double thymidine block at 10 mM for GFP stable cells and 5 mM for the importance of DGK activity in cell shape. However, it is not clear GFP-DGK KN cells for cell-cycle analysis. whether the difference in the cell size was due to the lack of DGK the At 4 h after washing out thymidine, 37% of the control cells tran- activity in the nucleus or in the cytoplasm because GFP-DGK KN sited to G /M phase, whereas the cells expressing GFP-DGK KN in expressed both in the cytoplasm and the nucleus. We, therefore, further G /M phase were only 17% (Fig. 7). 70% of the control cells were in examined the size of cells expressing DGK mutants only in the cytoplasm to G /M phase at 5 h, but it took 6–7 h for the population of GFP-DGK examine an effect of cytoplasmic DGK activity on the cell size. We used the KN in G /M phase to reach maximum. The GFP stable cells almost cells expressing GFP-DGK KNC1Am, because the mutants has no kinase returned to G phase at 7 h, and the size of population of them in G 1 1 activity and localizes only in the cytoplasm. On the other hand, the cells phase reached 60% at 8 h, the biggest value through this experiment. On expressing GFP-DGK C1B were used as the cells with cytoplasmic the other hand, 7 h after the release, 51% of GFP-DGK KN stable kinase activity (Figs. 2C and 3). No differences were found in the size of cells cells still remained in G /M phase, and only 14% cells were in G phase. 2 1 expressing GFP-DGK KNC1Am and GFP-DGK C1B; the size of cells Finally, a major population of GFP-DGK KN transited to G phase at expressing GFP-DGK C1B and KNC1Am were similar to that of GFP- 9 h; it was delayed by 2 h compared with the GFP stable cells. These or GFP-DGK-expressing cells (Fig. 5, left side). Furthermore, we measured results clearly reveal that the S phase of GFP-DGK KN was extended, the thickness of the CHO-K1 cells transiently expressing these proteins to suggesting that the extension causes enlargement of the cell. investigate whether overexpression of DGKKN flattened the cells and/or The disorder of cell cycle by nuclear expression of DGK KN sug- resulted in increase in cell volume. As shown in Fig. 5, the cells expressing gested that alteration of the cell cycle affects the intracellular localiza- GFP-DGK KN were slightly flatter than those of the cells expressing other tion of DGK. Therefore, we observed the localization of GFP-DGK in proteins. In addition, volume of the GFP-DGK KN cells was 1.36-fold; the stable cells after serum starvation for 24 h. Under control condi- the average of the diameter and thickness of GFP-DGK KN cells were 34.7 tions, the percentage of the stable cells expressing GFP-DGK both in m and 7.98 m, whereas those of the other cells were 28.86 m and 8.46 the cytoplasm and the nucleus was 34% and that expressing only in the m, respectively. These results indicated that the overexpression of DGK cytoplasm was 51% of all cells (Fig. 8A, control). The number of the cells KN affected the cell shape and volume. To confirm this, we further made expressing GFP-DGK both in the cytoplasm and the nucleus (type B) stable CHO-K1 cell lines of GFP, GFP-DGK, GFP-DGK KN, and GFP- increased from 35% to 54% by serum starvation. In contrast, the number DGK KNC1Am and compared the size of the cells. The stable cells of the cells expressing the fusion protein only in the cytoplasm (type A) expressing GFP-DGK KN in the nucleus and the cytoplasm were remark- decreased from 51% to 34%. The ratio of type B to type A increased from ably bigger than the others (Table 2). Importantly, the size of cells express- 0.692 to 1.557. These results indicate that GFP-DGK is transported ing GFP, GFP-DGK, and GFP-DGK KNC1Am were almost same. These from the cytoplasm to the nucleus under serum-starved conditions. results indicate that dominant negative effect on the nuclear DGK, but not However, these experiments were carried out under artificial condi- cytoplasmic DGK, influences the size and volume of cells. tions using exogenously expressed GFP-DGK. Therefore, to confirm Next, to elucidate the mechanism of DGK KN-induced enlargement nuclear transportation of endogenous DGK, we performed immuno- of cells, we compared proliferation of the stable cell lines (Fig. 6). Fig. 6 staining using anti-DGK antibody. In many control cells, endogenous shows that the DGKKN cells stated the division more slowly than the DGK was localized in the cytoplasm but not in the nucleus (Fig. 8B, others and that doubling time of GFP-DGKKN stable cells was 19.1 h, control). After 24-h serum starvation, DGK localized in the nucleus as which was calculated from its regression line. On the other hand, those of GFP, GFP-DGK, and GFP-DGKKNC1Am cells were 15.4, 15.8, much as in the cytoplasm (Fig. 8, serum starvation), indicating that, as well as GFP-DGK, endogenous DGK can be transported to the and 15.8 h. These results indicated that the proliferation rate of GFP- DGK KN was slightly slower than others. To directly investigate inhib- nucleus, and the nuclear transportation is induced by serum starvation. itory effect of nuclear DGK KN on the proliferation, we constructed These results, together with the inhibitory effect of DGKKN on cell CFP-DGK KN having nuclear localization signal (CFP-DGK KN- cycle, strongly suggest an important physiological function of nuclear NLS) and CFP-NLS as control and compared their proliferation rate. DGK in the cell cycle regulation. 6160 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK FIGURE 6. Effect of nuclear expression of DGK KN on the proliferation. A, proliferation of the CHO-K1 cells stably expressing GFP, GFP-DGK, GFP-DGK KN, and GFP-DGK KNC1Am. Each stable cell line was split into dishes at 0.2  10 cells/dish. Cells were harvested after 24-, 48-, and 72-h incubations and stained by 0.4% Trypan blue in PBS(). Then the numbers of living cells were counted. Each point and vertical bar indicates the mean  S.D. of six independent experiments. The regression coefficient of GFP-DGK KN was significantly different from those of the other three (p  0.001). B, live imaging of cell division of HeLa cells expressing CFP-NLS and CFP-DGKKN-NLS. The cell division of HeLa cells transiently expressing CFP- DGK KN NLS and CFP-NLS were observed as described under “Experimental Procedures.” Typical images of CFP-DGKKN-NLS and CFP-NLS cells are shown. Numbers on the right side show average of doubling time (n 25 for CFP-DGKKN-NLS, n 50 for CFP-NLS), which is from the first division to the second one. *, difference between the average of doubling time was significant (p  0.001). MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6161 Nuclear Transportation and Function of DGK FIGURE 7. Cell-cycle analysis of the stable cell lines of GFP, GFP-DGK KN, and GFP-DGKKN C1Am by flow cytometry. A, cell-cycle analysis of the stable cell lines of GFP, GFP-DGK KN, and GFP-DGKKN C1Am. 0.2  10 cells of each stable cell lines were spread on 10-cm dishes. The cells were synchronized by serum starvation for 24 h. After 24 h of serum restoration, the cells were treated with trypsin-EDTA and collected in a 1.5-ml tube. The collected cells were fixed by 70% EtOH and incubated with RNase A, and DNA was stained by propidium iodide. The cell cycle was analyzed by a FACSCalibur. B, cell-cycle analysis of the stable cell lines of GFP and GFP-DGK KN released from the double thymidine block. 0.2 10 cells of each stable cell line of GFP and GFP-DGKKN were spread on 10-cm dishes. The cells were synchronized at the beginning of the S phase with 10 (for GFP stable cells) or 5 mM (for GFP-DGK KN stable cells) thymidine. Every hour after the release, the cells were treated with trypsin-EDTA and collected in a 1.5-ml tube, and then subjected to flow cytometric analysis. DISCUSSION 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- In this report, we showed for the first time that DGK is transported DGK KNC1Bm versus GFP-DGK C1Bm in Fig. 3). These results suggest from the cytoplasm to the nucleus (Fig. 1). The nuclear transportation of that the kinase activity, although not essential, may have some roles in the DGK was independent of kinase activity of DGK (Fig. 3 and Table 1), but nuclear transportation of DGK. the mutant lacking kinase activity (DGK KN) showed much slower nuclear transportation than that of wild type. The mutation eliminating Instead of kinase activity, the C1 domain of DGK was essential for the kinase activity of GFP-DGK C1Am or GFP-DGK C1Bm made a remark- nuclear transportation of DGK. Namely, none of the C1 domain mutants 6162 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 Nuclear Transportation and Function of DGK (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 local- ized 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 flu- orescence 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 FIGURE 8. Effect of serum starvation on the localization of exogenous GFP-DGK inhibit the endogenous nuclear DGK by dominant-negative effects. and endogenous DGK in CHO-K1 cells. A, the change in subcellular localization of The cells expressing GFP-DGK KN were larger in the size (Fig. 5 and 5 GFP-DGK in the GFP-DGK stable cell line by serum starvation. 3.2  10 cells were spread onto glass bottom dishes and cultured overnight. The cells were further cultured Table 2), proliferated slowly (Fig. 6), and their S phase was extended for an additional 24 h in the presence (control) and absence of serum (free 24 h) and fixed. (Fig. 7), suggesting that enlargement of cell size is induced by protein Moreover, 100 cells were observed, and the number of the cells expressing GFP-DGK synthesis during extended S phase. In addition, serum starvation only in the cytoplasm (shaded bars), abundantly in the cytoplasm (), and equally in the cytoplasm and the nucleus (f) were counted. Same experiment was independently induced nuclear transportation of endogenous DGK and GFP-DGK performed three times and the averages  S.E. are plotted as percentage. Numbers stably expressing in the CHO-K1 cells (Fig. 8). These results strongly 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 implicate that DGK in the nucleus is involved in regulation of cell intact cells under serum starvation conditions. CHO-K1 cells were spread onto glass cycle. bottom dishes and cultured overnight. After being cultured for an additional 24 h in the Involvement of DGK in cell-cycle regulation is supported by several 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- reports as follows. First, inhibition of DGK activity prevents transition DGK antibody. from G to S phase (39). Second, the involvement of other DGK sub- types such as DGK in the cell cycle has also been reported, although confirmed the expression of DGK in various tissues, including kidney, there are some differences; COS-7 cells expressing DGK showed the muscle, and other tissues, in addition to brain (supplemental Fig. S2). increase in the size of G cell population and decrease in that of G /M Therefore, at least in these cells and tissues, it is expected that nuclear 1 2 cell population but no effects of DGK KN on the cell cycle were DGK is involved in cell-cycle regulation. In fact, regulation of differ- detected (21). The differences may be due to subtype specificity and/or entiation by DGK in HL-60 cells has been suggested (41, 43). different cell types used. However, it still remains to be solved how DGK can regulate the cell Both DGK and DGK are abundantly expressed in the brain (40), cycle. The amount of nuclear phosphatidylinositols, including phos- although it is unclear about their functions as a regulator of the cell cycle phatidylinositol 4,5-bisphosphate, fluctuates during the cell cycle (22), in the central nervous system. However, expression of DGK has been and the DG mass in the nucleus increases in G /M phase (27). Further- recently reported in HL-60, U937, and NIH3T3 cells (41–43). We also more, IIPKC translocates into the nucleus and phosphorylates lamin B MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6163 Nuclear Transportation and Function of DGK (1996) J. Biol. Chem. 271, 10230–10236 in the G /M phase (44). These reports suggest that the DG derived from 18. Ding, L., Traer, E., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1998) phosphatidylinositol 4,5-bisphosphate recruits PKC to regulate G /M J. Biol. Chem. 273, 32746–32752 phase. In other words, regulation of nuclear DG mass is important for 19. Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, cell-cycle regulation, and DGK might be involved in the control of M., Takenawa, T., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422–10428 20. Bregoli, L., Baldassare, J. J., and Raben, D. M. (2001) J. Biol. Chem. 276, 23288–23295 nuclear DG. Alternatively, PA produced by DGK may have some func- 21. Topham, M. K., Bunting, M., Zimmerman, G. A., McIntyre, T. M., Blackshear, P. J., tion in cell-cycle regulation. In fact, the amount of PA in the nucleus also and Prescott, S. M. (1998) Nature 394, 697–700 changes by the stimulation of -thrombin, which has a mitogenic effect 22. York, J. D., and Majerus, P. W. (1994) J. Biol. Chem. 269, 7847–7850 (45). Accordingly, the mechanism of cell-cycle regulation by DGK is an 23. D’Santos, C. S., Clarke, J. H., and Divecha, N. (1998) Biochim. Biophys. Acta 1436, 201–232 important issue to be solved next. 24. Divecha, N., Banfic, H., and Irvine, R. F. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Mar 10, 2006

References