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c-Abl is a tyrosine kinase involved in many cellular processes, including cell cycle control and proliferation. However, little is known about its substrates. Here, we show that c-Abl directly phosphorylates diacylglycerol kinase α (DGKα), an important regulator of many cellular events through its conversion of diacylglycerol to phosphatidic acid. We found that DGKα was transported from the cytoplasm to the nucleus in response to serum starvation, and serum restoration induced the nuclear export of the enzyme to the cytoplasm. This serum-induced export involves two tyrosine kinases, c-Src and c-Abl. The latter, c-Abl, is activated by c-Src, phosphorylates DGKα, and shuttles between the nucleus and the cytoplasm in a direction opposite to that of DGKα in response to serum restoration. Moreover, an in vitro phosphorylation assay using purified mutants of DGKα identified Tyr-218 as a site of phosphorylation by c-Abl. We confirmed these results for endogenous DGKα using an antibody specific for phospho-Tyr-218, and this phosphorylation was necessary for the serum-induced export of DGKα. These results demonstrate that the nucleo-cytoplasmic shuttling of DGKα is orchestrated by tyrosine phosphorylation by the Src-activated tyrosine kinase c-Abl and that this phosphorylation is important for regulating the function of cytoplasmic and/or nuclear DGKα.
Cellular activities are controlled by various substances, including lipids. Lipids are the principal components not only of the cytoplasmic membrane but also of the membrane structure of organelles, including the mitochondria, nucleus, endoplasmic reticulum, and Golgi apparatus. In addition, lipids have important roles in signal transduction. The cytoplasmic membrane contains phosphatidylinositol 4,5-bisphosphate, which is hydrolyzed to diacylglycerol (DG)3 and inositol 1,4,5-trisphosphate by phospholipase Cβ upon stimulation. DG activates protein kinase C (PKC) in cooperation with Ca2+ release induced by inositol 1,4,5-trisphosphate. Thus, DG is an important lipid messenger in signal transduction.
Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA) (1), resulting in an attenuation of PKC activity. PA is also an activator for many other kinases, including phosphatidylinositol 4-phosphate 5-kinase (2, 3), protein kinase Cζ (4), and the mammalian target of rapamycin (5). Therefore, DGK is thought to be one of the key enzymes in lipid signal transduction. Mammalian DGKs have 10 subtypes and are categorized into five groups, depending on their primary structure (6). All DGKs, except DGKθ, have two cysteine-rich regions (C1A and C1B domains) in the regulatory domain at the N terminus and a catalytic domain at the C terminus. DGKθ has three C1 domains, and catalytic domains of type II DGKs are separated. In addition to these domains, they have different structures, depending on their groups. Type I DGKs, DGKα, -β, and -γ, have an EF-hand motif and a recoverin homology (RVH) domain. Type II DGKs, DGKδ, -η, and -κ, have a pleckstrin homology domain instead of an EF-hand and RVH domain at the N terminus. DGKδ and -η have a sterile α motif domain at the C terminus. DGKκ has an EPAP repeat at the N terminus and a PDZ binding domain at the C terminus. DGKϵ is a Type III DGK, and it contains only a C1 domain. Type IV DGKs, DGKζ and -ι, have a myristoylated alanine-rich protein kinase C substrate phosphorylation site-like region at the N terminus and four ankyrin repeats at the C terminus. Finally, DGKθ, which is a Type V DGK, has a proline- and glycine-rich domain and a pleckstrin homology domain with an overlapping Ras association domain. DGKs are thought to be involved in development, differentiation, construction of neural networks, and immunity (7). However, subtype-specific functions and regulatory mechanisms remain poorly characterized.
Some DGKs show translocation to the plasma membrane, where DG is produced, in response to various stimulations (8–10). However, lipid metabolism also exists in the nucleus, and the production of DG and PA in the nucleus has been reported (11–13). Moreover, it was reported that DGKζ, -θ, and -γ were localized in the nucleus (14–16). These findings suggest that DGKs are important for nuclear DG/PA metabolism.
In a series of experiments aimed at elucidating the subtype-specific functions of DGKs, we found that DGKα shuttles between the cytoplasm and the nucleus. However, DGKα does not have a conserved nuclear localization signal or a nuclear export signal. Therefore, we tried to clarify the mechanism of DGKα nucleo-cytoplasmic shuttling and found that the nuclear export of the enzyme is regulated by phosphorylation at Tyr-218 by C-Abl.
NIH3T3 cells and COS-7 cells were purchased from the RIKEN Cell Bank. DDT1-MF2 (DTT) cells were obtained from B. B. Fredholm (Karolinska Institute, Stockholm, Sweden). The plasmids encoding either wild-type Src or a kinase-negative Src C-terminally fused to a FLAG tag were obtained from Dr. Fukami (Kobe University, Kobe, Japan). The plasmids encoding either wild-type c-Abl or a kinase negative c-Abl were kindly provided by Dr. Tanaka (Hokkaido University, Sapporo, Japan). The anti-GFP antibodies were made in house, and the anti-DGKα antibodies were described previously (18). We obtained mouse anti-lamin A/C antibody, mouse anti-Src antibody, and mouse anti-Tyr(P) antibody (4G10) from Upstate Biotechnology, Inc. (Lake Placid, NY), rabbit anti-c-Abl antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), mouse anti-Lck from BD Transduction Laboratories (San Jose, CA), rabbit anti-Tyr(P)-412-c-Abl antibody from IMGENEX (San Diego, CA), rabbit anti-FLAG antibody from Affinity Bioreagents (Golden, CO), and rabbit anti-MBP antibody from DIATHEVA (Viale Piceno, Fano, Italy) as primary antibodies. We purchased peroxidase-labeled goat anti-rabbit and mouse IgG from Jackson ImmunoResearch Laboratories (West Grove, PA) and peroxidase-labeled goat anti-guinea pig IgG from Chemicon International (Temecula, CA) as secondary antibodies. DGKα siRNA from Santa Cruz Biotechnology, Inc. was donated by Dr. Yamashita and Dr. Akifusa (Kyusyu University, Fukuoka, Japan) (19).
COS-7 and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (Nacalitesque, Kyoto, Japan). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. All media contained 25 mm glucose, and they were all buffered with 44 mm NaHCO3 and supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Invitrogen). The fetal bovine serum used was not heat-inactivated.
The constructs encoding DGKα N-terminally fused to GFP (GFP-DGKα) were described previously (8). To construct the plasmid encoding DGKα with the substitution of Phe for Tyr-218 (Y218F), mutagenesis was carried out as described by the manufacturer's protocol using a QuikChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA). The primers for Y218F were 5′-AGACCCAAGAGGTTCCCCAGGCCTGTCTTCTGCAACCTGTGCGAGTCG-3′ and 5′-CGACTCGCACAGGTTGCAGAAGACAGGCCTGGGGAACCTCTTGGGTCTC-3′. The mutation was confirmed by verifying its sequence.
cDNA fragments of either wild-type or kinase-negative c-Abl with a HindIII site at the N terminus and a KpnI site at the C terminus were produced by PCR using mouse c-Abl cDNA as the template. The PCR products were first subcloned into a pUC118 vector (Takara, Shiga, Japan). After digestion with HindIII and KpnI, the cDNA encoding each fragment was subcloned into the HindIII and KpnI site in the pEGFP-C1 vector or the pDsRed-Monomer-C1 vector (Clontech). The primers used were 5′-TTAAGCTTCGGCCACCATGGGGCAGCCTGGA-3′ and 5′-TTGGTACCGTCCTCCGGACAATGTCGCT-3′.
NIH3T3 cells (7.5 × 104 cells/dish) grown on a glass bottom dish (MatTek Corp., Ashland, MA) were transfected using 3 μl of FuGENETM6 Transfection reagent (Roche Applied Science) and 1 μg of DNA according to the manufacturer's protocol. Transfected cells were cultured at 37 °C for about 24 h before use.
Cy3 and GFP fluorescence was observed under a confocal laser scanning fluorescent microscope (Carl Zeiss, Jena, Germany). The GFP fluorescence was monitored at 488 nm using an argon laser for excitation with a 515-nm-long pass barrier filter. Cy3 fluorescence was monitored at 543 nm using a HeNe 1 laser for excitation with a 560–615-nm band pass filter.
Quantitation of the subcellular distribution of the GFP fusion proteins was defined as described below. The cells in which the ratio of the GFP fluorescence intensity of the cytoplasm and the nucleus was under 0.5 were defined as “only in the cytoplasm,” and the cells in which the ratio was 0.5–0.7 were defined as “abundantly in the cytoplasm.” If the ratio was over 0.7, the cells were defined as “equally in the cytoplasm and the nucleus.”
Fractionation was performed as described previously (20). NIH3T3 or DDT cells were harvested and resuspended in cytoplasmic lysis buffer containing 10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm sodium orthovanadate. Cells were incubated on ice for 15 min. Nonidet P-40 was added to a final concentration of 0.4%. Cells were vortexed vigorously for 10 s and then centrifuged at 18,000 × g for 30 s. The supernatant was kept as the cytoplasmic fraction. The pellet was washed twice in lysis buffer containing 0.2% Nonidet P-40 and resuspended in nuclear lysis buffer (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride). The tube was vigorously rotated at 4 °C for 15 min and centrifuged for 5 min at 18,000 × g. The supernatant was designated as the nuclear fraction. Nuclear fractions were subjected to immunoblotting.
The lysates from COS-7 cells transfected with wild-type or kinase-negative GFP-c-Abl were incubated with rotation with an anti-GFP antibody immobilized on protein A-Sepharose beads in homogenizing buffer without Triton-X 100 at 4 °C for 2 h. After incubation, the protein A-Sepharose beads were washed with c-Abl kinase buffer three times.
Plasmids (~32 μg) were electroporated into COS-7 cells using a gene pulser (Bio-Rad, 975 microfarads, 220 mV) or transfected into NIH3T3 cells using the FuGENETM6 transfection reagent. After being cultured, the cells were harvested and centrifuged at 5,500 × g for 3 min. The cells were resuspended in homogenizing buffer (250 mm sucrose, 10 mm EGTA, 2 mm EDTA, 50 mm Tris-HCl, 200 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, pH 7.4, 1 mm NaF, 1 mm sodium vanadate) and sonicated (UD-210 TOMY, Tokyo, Japan; output 3, 15 s, two times).
For immunoblotting, the samples were subjected to 7.5% SDS-PAGE, followed by blotting onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Nonspecific binding sites were blocked by incubation with 5% skim milk in 0.01 m PBS containing 0.03% Triton X-100 (PBS-T) at 4 °C overnight. The membrane was incubated with primary antibody for 1 h at room temperature. After washing with PBS-T, the membrane was incubated with secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. After three rinses with PBS-T, the immunoreactive bands were visualized using a chemiluminescence detection kit (ECL, Amersham Biosciences).
For the preparation of the anti-phospho-Tyr-218 of DGKα antibody, an oligopeptide corresponding to amino acids 213–223 of pig DGKα containing phospho-Tyr-218 (FPRPVpYVNLCE) was used as an antigen. The synthetic peptide was conjugated to keyhole limpet hemocyanin, and 20 μg of keyhole limpet hemocyanin-conjugated antigen was emulsified and injected into a 10-week-old female guinea pig. Booster injections (20 μg each) were given at 2-week intervals. Three days after the sixth boost, serum was collected and purified with an affinity column using the antigen peptide.
cDNA fragments of DGKα fragments with an EcoRI site at the N terminus and a SalI site at the C terminus were produced by PCR using pig DGKα cDNA as the template. The PCR products were first subcloned into a pUC118 vector (TAKARA, Shiga, Japan). After digestion with EcoRI and SalI, the cDNA encoding each fragment was subcloned into the EcoRI and SalI sites in the pMAL-C2 vector (New England BioLabs, Ipswich, MA). The primers for the DGKα regulatory domain (bp 1–1116) were 5′-TTGAATTCATGTCCAAGGAGAGGGGG-3′ and 5′-TTGTCGACTCAGTTAGAAACGGGGTC-3′. The primers for RDΔhinge (318 aa) (bp 1–954), for RVH-EF-C1 domains (252 aa) (bp 1–756), for RVH-EF-C1a (204 aa) (bp 1–612), and for RVH-EF hand (112 aa) (bp 1–336) were 5′-TTGTCGACTCAACACTCATGGCCCATGGC-3′, 5′-TTGTCGACTCAAACACAGGGCAGGGCCTTCAT-3′, 5′-TTGTCGACTCACTGCCCATTGTCCTTCAA-3′, and 5′-TTGTCGACTCAGTCTTCTGGCCGGCCGCC-3′, respectively.
DH5α cells were transformed with expression plasmids for MBP fusion proteins. MBP fusion proteins were expressed and purified according to the manufacturer's protocols (New England Biolabs). Expression of MBP fusion proteins was induced with 0.3 mm IPTG at 25 °C for 16 h. The induced cells were harvested and sonicated in column buffer (20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DDT, 200 mm NaCl, 1% Triton X-100, 20 μg/ml leupeptin, 1 mm PMSF). After centrifugation at 10,000 × g for 30 min, MBP fusion proteins were applied to amyrose resin and incubated overnight at 4 °C. After washing with column buffer, to cut MBP, 10× factor Xa cleavage buffer and factor Xa were added and incubated overnight at 4 °C. Eluate was dialyzed against PBS(−).
Purified proteins were incubated in c-Abl kinase buffer (50 mm HEPES, pH 7.4, 10 mm MgCl2, 10 mm MnCl2, 2 mm DDT, 0.1 mm sodium vanadate) with the precipitants obtained from the immunoprecipitation using an anti-GFP antibody and [γ-32P]ATP (MP Biomedicals, Solon, OH) for 15 min at 30 °C. Reaction products were subjected to SDS-PAGE and analyzed using BSA2500 (Fujifilm, Tokyo, Japan).
To specifically detect the cells transfected with c-Abl siRNA, we employed thymidine dimer methods described previously (21). Briefly, 30 μg of siRNA was irradiated with 2.0 J/cm2 UV. The irradiated siRNA (300 nm) and 1 μg of the plasmid encoding GFP-DGKα were simultaneously transfected into 4 × 104 NIH3T3 cells. Twenty-four hours after the transfection, serum was depleted for 24 h, and then serum was added and incubated for 3 h. Before and after serum starvation and 3 h after serum restoration, the cells were fixed. After immunostaining with anti-thymidine dimer (Kyowa Medics), the localization of GFP-DGKα in the thymidine-dimer positive cells was categorized into three groups and plotted.
When GFP-DGKα was transfected into NIH3T3 cells, it localized mainly in the cytoplasm but not in the nucleus (Fig. 1A, top). However, the percentage of the cells expressing GFP-DGKα both in the cytoplasm and in the nucleus gradually increased over a several-day course of culturing (Fig. 1A, bottom). At day 1 (24 h after transfection), the percentage of cells expressing GFP-DGKα only in the cytoplasm was 56% (Fig. 1B), which decreased to 28% at Day 3 (72 h after transfection). In contrast, the percentage of cells expressing GFP-DGKα equally in the cytoplasm and the nucleus increased from 31 to 52% within 3 days (Fig. 1B). To quantify the nuclear localization of the enzyme, the ratio of the cells expressing GFP-DGKα equally in the cytoplasm and in the nucleus to those cells expressing the protein only in the cytoplasm was defined as the nuclear localization index, which is plotted in Fig. 1C. The index increased from 0.55 at day 1 to 1.36 at day 3, indicating that GFP-DGKα was transported from the cytoplasm to the nucleus in a time-dependent manner. The fact that this nuclear transportation took 2–3 days suggests the lack or shortage of some factor(s) from the medium required for triggering nuclear import. Therefore, we determined the effect of serum depletion on nuclear import. Serum starvation accelerated the rate of the nuclear transportation; significant nuclear transportation was detected within 24 h after serum depletion (Fig. 1D).
Interestingly, the subsequent restoration of serum induced the export of DGKα from the nucleus to the cytoplasm. The nuclear localization index increased from 0.40 to 1.93 during 24 h of serum starvation but reversed to 0.53 within 3 h of serum addition (Fig. 1E), indicating that DGKα shuttles between the cytoplasm and nucleus, depending on the culture conditions. The serum-dependent shuttling of DGKα was also detected in DDT cells derived from Syrian hamster leiomyosarcoma (Fig. 1F), indicating that this phenomenon is not unique to NIH3T3 cells. Furthermore, biochemical fractionation followed by immunoblotting revealed that endogenous DGKα also shuttles between the cytoplasm and the nucleus in a serum-dependent manner (Fig. 1G).
To identify the factor(s) involved in the serum-induced nuclear export of DGKα, we tested several growth factors instead of serum. Among them, platelet-derived growth factor (PDGF) similarly induced the nuclear export (Fig. 1H), suggesting the involvement of tyrosine kinases in this process. Previous reports showed that tyrosine phosphorylation by Src family protein-tyrosine kinases is required for the recruitment of DGKα to the membrane (12). Therefore, we investigated the effect of a tyrosine kinase inhibitor, herbimycin, on the serum-induced nuclear export of DGKα. The herbimycin treatment significantly inhibited the movement of DGKα from the nucleus to the cytoplasm (Fig. 2A). Next, we checked for the presence of Lck and/or c-Src in the NIH3T3 cells because it has been reported that c-Src and Lck directly phosphorylate DGKα (9, 10). The cells endogenously express c-Src but not Lck (Fig. 2B), suggesting that c-Src is a possible candidate. However, GFP-tagged c-Src mainly localized in the cytoplasm under all of the conditions we tested (data not shown), suggesting that the serum-induced nuclear export of DGKα involves an additional factor, which is regulated by c-Src and phosphorylates DGKα in the nucleus.
c-Abl tyrosine kinase was an attractive candidate as an additional factor regulating the nuclear export of DGKα because it is activated by Src (21, 22) and exhibits nucleo-cytoplasmic shuttling (23). Therefore, we checked if NIH3T3 cells endogenously express c-Abl, and we determined the effects of a specific c-Abl inhibitor, STI571, on the serum-induced nuclear exportation of DGKα. c-Abl is endogenously expressed in NIH3T3 cells (Fig. 2B), and STI571 inhibited the nuclear export of GFP-DGKα in a dose-dependent manner (Fig. 2, C and D), suggesting the involvement of c-Abl. To confirm it, we used the irradiated siRNA of c-Abl, as described under “Methods.” First we checked whether endogenous c-Abl was down-regulated by the irradiated siRNA treatment. Fig. 2E indicates that endogenous c-Abl was down-regulated in the cells transfected with siRNA, which was recognized by anti-T-T dimer antibody. We then investigated the localization of GFP-DGKα in the T-T-positive cells. As shown in Fig. 2F, the serum-induced nuclear exportation of DGKα was significantly inhibited by the c-Abl siRNA treatment, confirming the importance of the enzyme for the exportation.
Next, we investigated the shuttling of c-Abl in response to serum. GFP-c-Abl showed nucleo-cytoplasmic shuttling in response to serum, but the direction was opposite that of DGKα; GFP-c-Abl was exported from the nucleus to the cytoplasm by serum depletion, whereas it was imported to the nucleus by serum restoration (Fig. 3A), fitting with our hypothesis that c-Abl directly regulates the serum-induced nuclear export of DGKα. Furthermore, co-expression of mDsRed-tagged c-Abl enhanced the cytoplasmic localization of GFP-DGKα before serum depletion (Fig. 3B); co-expression of c-Abl increased the percentage of cells expressing GFP-DGKα only in the cytoplasm from 60 to 80% and decreased that of the cells both in the cytoplasm and the nucleus from 30 to 15%. In addition, co-expression of c-Abl inhibited the serum depletion-induced nuclear import of DGKα; the nuclear index was lower than 1 in the presence of c-Abl (Fig. 3B, bottom). Together, these results suggest that growth factors, including PDGF, in the serum activate c-Src and that activated c-Src phosphorylates c-Abl, resulting in the activation and nuclear import of c-Abl, and finally, c-Abl phosphorylates DGKα in the nucleus to export DGKα.
To confirm this hypothesis, we tried to detect the c-Src-dependent activation of c-Abl using an anti-phospho-Tyr-412-c-Abl antibody, because the phosphorylation of Tyr-412 is a marker for the activation of c-Abl (21, 22). In these experiments, we employed a kinase-negative c-Abl (c-Abl KN) to avoid autophosphorylation. When co-transfected with Src-FLAG, the tyrosine phosphorylation at 412 on GFP-c-Abl KN was enhanced by H2O2 stimulation, which is an activator of c-Src (Fig. 4A). Serum restoration induced the tyrosine phosphorylation of GFP-c-Abl KN only when Src-FLAG was co-expressed (Fig. 4B) and tyrosine phosphorylation was inhibited by herbimycin (Fig. 4B), indicating that serum-activated c-Src phosphorylates c-Abl in NIH3T3 cells. Next, we investigated whether c-Abl phosphorylates DGKα in NIH3T3 cells under serum restoration. Serum treatment on the NIH3T3 cells overexpressing c-Abl enhanced tyrosine phosphorylation of endogenous DGKα, but the enhancement was not detected in the case of the KN c-Abl (Fig. 4C). Importantly, the serum-induced enhancement of tyrosine phosphorylation was inhibited by STI571 (Fig. 4C), indicating that DGKα is tyrosine-phosphorylated by c-Abl, which is activated in a serum-dependent manner.
To see the direct phosphorylation of DGKα by c-Abl, an in vitro kinase assay was performed. Incubation of purified MBP fused to the regulatory domain (RD) of DGKα with WT GFP-c-Abl, which was immunoprecipitated from COS-7 cell lysates by an anti-GFP antibody, caused the phosphorylation of MBP-DGKαRD (Fig. 4D). In contrast, GFP-c-Abl KN did not phosphorylate the MPB-RD. These results demonstrate that c-Abl directly phosphorylates the RD of DGKα. Together, these results support the hypothesis that the c-Src/c-Abl cascade is involved in the serum-induced nuclear export of DGKα.
To identify the phosphorylation site in DGKα, we produced a series of mutated RD domains fused with MBP. After cutting out MBP with factor Xa (Novagen), we performed an in vitro phosphorylation assay using the purified proteins and v-Abl (New England BioLabs), which has a catalytic region homologous to c-Abl.
Again, direct phosphorylation of RD by v-Abl was confirmed (Fig. 5A). Even when the hinge region and C1b were truncated, the strong phosphorylation signals were still detected (see 252 in Fig. 5A). However, deletion of the C1a domain resulted in a significant reduction of phosphorylation (Fig. 5A), indicating that there is, at least, a phosphorylation site in the C1a domain. Because there are two tyrosines in the C1a region (Tyr-218 and -240), we produced mutants in which Tyr-218 and -240 were substituted with Phe and performed an in vitro phosphorylation assay. Y240F, but not Y218F, was phosphorylated by v-Abl (Fig. 5B), indicating that v-Abl directly phosphorylates Tyr-218 of DGKα in vitro. Fig. 5 also shows that there is an additional phosphorylation site in the EF-hand domain because 204 showed a signal that disappeared when the EF-hand was further deleted. However, we could not identify the phosphorylation sites in the EF-hand (data not shown).
To confirm that endogenous DGKα is phosphorylated by c-Abl in vivo, we produced an antibody specific for phospho-Tyr-218. The antibody recognized the phosphopeptide, including Tyr(P)-218 (pY218), but not the control phosphopeptide (CQKYMEpYSTKKV) or Tyr(P)-218 after treatment with λ-phosphatase (Fig. 6A). To further confirm the specificity of the antibody, peptide 252 (WT) and its YF mutant at 218 (YF) were employed. The peptides were phosphorylated by v-Abl in vitro and applied to SDS-PAGE followed by Western blotting. The Tyr(P)-218 antibody recognized the phosphorylated WT 252 but not the YF mutant (Fig. 6B). Next, we performed Western blotting using this antibody, to investigate whether endogenous DGKα is really phosphorylated at Tyr-218 and where the phosphorylation occurs. Fig. 6C shows that cytosolic DGKα was phosphorylated at Tyr-218, indicating that Tyr-218 is phosphorylated in vivo. However, we did not detect phospho-Tyr-218 DGKα in the nuclear fraction. Moreover, to confirm that the phosphorylation at 218 was dependent on c-Abl, we tested the effect of STI571 on the phosphorylation. The Tyr-218 phosphorylation was inhibited by STI571 (Fig. 6D).
Finally, to confirm that phosphorylation of Tyr-218 is necessary for the serum-induced export of DGKα, we checked the shuttling of the GFP-Y218F mutant, which is a GFP fusion of the full-length DGKα with a Phe substitution for Tyr-218. The GFP-Y218F mutant showed nuclear import similar to WT upon serum depletion, but it could not be exported from the nucleus in response to the addition of serum (Fig. 6E), indicating that phosphorylation of Tyr-218 is necessary for the serum-induced export.
This is the first report of the nucleo-cytoplasmic shuttling of DGKα under serum starvation and restoration (Fig. 1). The serum-induced export of DGKα is regulated by the sequential activation of c-Src and c-Abl. This is supported by the following results: herbimycin, STI571, and siRNA knockdown of c-Abl inhibited the serum-induced export (Fig. 2); c-Abl was phosphorylated by c-Src in response to serum (Fig. 4B); DGKα was phosphorylated by c-Abl in vivo and in vitro (Fig. 4, C and D); c-Abl showed the same serum-dependent nucleo-cytoplasmic shuttling but in the direction opposite that of DGKα (Fig. 3A); and co-expression of c-Abl prevented the serum-induced nuclear import of DGKα (Fig. 3B). In addition, Tyr-218 was identified as a phosphorylation site for c-Abl (Figs. 5 and and6),6), and this phosphorylation is important for the serum-induced export of DGKα (Fig. 6E).
These results suggest that DGKα is an important substrate of c-Abl. The tyrosine kinase was originally identified as the cellular homolog of the v-Abl oncogene product of the Abelson murine leukemia virus (24, 25). Subsequently, c-Abl was shown to be involved in human leukemia as a result of chromosomal translocation events that fuse the bcr and c-abl genes, producing the Bcr-Abl chimeric oncogene (26, 27). It has been reported that the normal c-Abl protein has a role related to the cell cycle, but little is known about its physiological roles and its substrates (28). Together with our results, these facts suggest an important correlation between the shuttling of DGKα and the function of c-Abl. However, discerning the physiological function of DGKα is beyond the scope of this paper. One possible speculation is that cytoplasmic DGKα positively regulates cell proliferation, and it may be pooled in the nucleus under non-proliferative conditions, because serum depletion induced the nuclear transportation of DGKα, and serum restoration caused its nuclear export. This fits with the facts that the hyperproliferative cell line K562 possesses a form of Bcr/Abl with constitutive activity, and that a DGK inhibitor prevents the proliferation of K562 cells (data not shown). Alternatively, to keep cells non-proliferative, nuclear DGKα may cause cells to arrest in G1, similar to DGKζ (29), or keep the cells in the G0 phase. Indeed, a stable line of KN DGKα displayed an abnormal cell cycle (data not shown), and the DGK inhibitor prevented the G0 to G1 transition in NIH3T3 cells following release from serum starvation (data not shown). This hypothesis fits the previous report that c-Abl is involved in S phase entry (30) and that nuclear levels of DG and PA fluctuate during the cell cycle (11–13, 31). Furthermore, DGKα may participate in nuclear membrane lipid metabolism because the nuclear membrane obviously contains many kinds of lipids, and DGKα is translocated to the nuclear matrix (32). In speculating about the physiological functions of nuclear DGKα, we have to be careful because treatment with DGK inhibitors and overexpression of KN DGKα affect both cytosolic and nuclear DGK activities, and cytosolic and nuclear DGKα may have opposing functions.
How can the phosphorylation at Tyr-218 regulate the export of DGKα? The phosphorylation at this site may inhibit the nuclear localization signal function of the C1 domain because Tyr-218 is in the C1 domain, because we previously reported that the C1 domain of DGKγ has an important role in nuclear transport (17). Binding to importin-α, a carrier protein that transports some proteins to the nucleus (33–35), or to an as yet unidentified carrier protein may be regulated by this phosphorylation. Alternatively, the phosphorylation may regulate the interaction between the C1 domain and the N-terminal region of DGKα, because the N-terminal region, including the RVH domain and the EF-hand motif, functions as a nuclear export signal (data not shown); the RD was localized in the cytoplasm, although it contains the C1 domain. Another possibility is that the phosphorylation affects the lipid-binding ability of the C1 domain because it is a well known lipid-binding domain, and other proteins, including PKC, c-Raf and Chimaerin, also have the C1 domain (36). To reveal the precise mechanism, further research is needed.
Needless to say, there may be additional phosphorylation sites recognized by c-Abl. For example, there seems to be, at least, a c-Abl phosphorylation site in the EF-hand. Furthermore, there may be more phosphorylation sites in catalytic domains because we did not address them in this study. In addition, it has been reported that Tyr-334 is phosphorylated by c-Src and Lyn (9, 10, 37). Interestingly, Tyr-334 phosphorylation is involved in the plasma membrane accumulation of DGKα that occurs in response to several stimulations (9, 10, 37), whereas Tyr-218 phosphorylation is necessary for the serum-induced export of DGKα from the nucleus to the cytoplasm. These results indicate that phosphorylation of different tyrosine residues leads to different directions of translocation. Such a mechanism could be important for DGKα activity. These results suggest that DGKα can change its binding partner, depending on the combination of multiple phosphorylation events, with different translocation outcomes.
Finally, our model for DGKα nucleo-cytoplasmic shuttling is shown in Fig. 7. DGKα is localized in the cytoplasm under normal conditions, where the C1 domain, the putative nuclear transportation signal, is blocked by the N-terminal region. Under serum starvation, DGKα is transported to the nucleus because the C1 domain gets exposed. The trigger for such a conformational change is unknown, but dephosphorylation may be a key step. Serum restoration induces the sequential activation of c-Src and c-Abl. The activated c-Abl is transported to the nucleus and phosphorylates DGKα at residue 218, although we did not succeed in detecting phospho-Tyr-218 in the nucleus. This may be because once that tyrosine is phosphorylated, DGKα is immediately exported to the cytoplasm. However, our results cannot completely rule out the possibility that c-Abl phosphorylates DGKα in cytoplasm. In conclusion, the well designed nucleo-cytoplasmic shutting system could contribute to the function of DGKα in the nucleus and/or the cytoplasm.
*This work was supported in part by a Grant-in-aid for Scientific Research (C) and the Global Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
3The abbreviations used are: