The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses. Therefore, p53 protein levels and activity need to be kept under tight control. We report here that diacylglycerol kinase ζ (DGKζ) binds to p53 and modulates its function both in the cytoplasm and nucleus. DGKζ, a member of the DGK family that metabolizes a lipid second messenger diacylglycerol, localizes primarily to the nucleus in various cell types. Recently, reports have described that excitotoxic stress induces DGKζ nucleocytoplasmic translocation in hippocampal neurons. In the study reported here we found that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity against doxorubicin-induced DNA damage by facilitating cytoplasmic anchoring and degradation of p53 through a ubiquitin–proteasome system. Concomitantly, decreased levels of nuclear DGKζ engender downregulation of p53 transcriptional activity. Consistent with these in vitro cellular experiments, DGKζ-deficient brain exhibits high levels of p53 protein after kainate-induced seizures and even under normal conditions. These findings provide novel insights into the regulation of p53 function and suggest that DGKζ serves as a sentinel to control p53 function both during normal homeostasis and in stress responses.

The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses such as DNA-damage, hypoxia, hyperproliferation, and oncogene activation. In response to various stresses, p53 triggers various cellular reactions that engender DNA repair, cell cycle arrest, apoptosis, senescence, differentiation, and inhibition of angiogenesis (Polager and Ginsberg, 2009; Teodoro et al., 2007; Vousden, 2006). Approximately 50% of human cancers bear p53 gene mutations. In most of the remaining cancer cases, p53 activity is compromised by alternative mechanisms (Vogelstein et al., 2000). In addition, activation of p53 is implicated in the apoptotic cell death of post-mitotic neurons after stroke, traumatic brain injury, and neurodegenerative diseases (Culmsee and Mattson, 2005). Hippocampal neurons of p53 knockout mice are prevented from cell death in the kainate-induced seizure model (Morrison et al., 1996) and primary cultured hippocampal or cortical neurons derived from p53-deficient mice exhibit decreased sensitivity to excitotoxicity caused by kainate and glutamate (Xiang et al., 1996). These results of studies suggest that p53 plays a key role in the neuronal death process in response to various stresses.

In pathophysiological signaling, phosphoinositide (PI) turnover produces a well-known lipid messenger, diacylglycerol (DG), in response to external stimuli (Cockcroft and Thomas, 1992; Divecha and Irvine, 1995). DG acts as an activator of various effector proteins containing DG-responsive C1 domain, including protein kinase C (PKC), Ras guanyl nucleotide releasing proteins (RasGRP), PKDs, Munc-13s and transient receptor potential (TRP) (Hardie, 2003; Topham, 2006). Sustained activation of the DG pathway would otherwise promote a maladaptation such as dysregulated proliferation or necrosis/apoptosis (Nishizuka, 1984). Therefore, levels of DG must be controlled strictly to keep the cellular environment within a physiological range. In addition, phosphatidic acid (PA) has been shown to regulate several proteins such as a mammalian target of rapamycin (mTOR), Ras guanosine triphosphatase (RasGTPase)-activating proteins (RasGAPs), phosphatidylinositol 4P 5-kinase (PI4P5K), p47phox and son-of-sevenless (Sos), a Ras guanine nucleotide exchanging factor (RasGEF) (Zhang and Du, 2009). Therefore, diacylglycerol kinase (DGK), an enzyme that phosphorylates DG to PA, is involved in various pathophysiological cellular responses through the conversion of DG to PA (Kanoh et al., 1990; van Blitterswijk and Houssa, 1999). It can be regarded as the regulator of DG/PA signaling.

DGK consists of a family of isozymes, each of which has a unique character in terms of regulatory mechanism, binding partner, and subcellular localization (Goto et al., 2007; Mérida et al., 2008; Sakane et al., 2007; Topham and Epand, 2009). Of the isozymes, DGKζ, containing a nuclear localization signal (NLS), localizes to the nucleus in cells of various kinds (Bunting et al., 1996; Goto and Kondo, 1996; Hozumi et al., 2003; Katagiri et al., 2005). Under pathological conditions, DGKζ is shown to translocate from the nucleus to the cytoplasm in hippocampal neurons in animal models of ischemia (Ali et al., 2004) and kainate-induced seizures (Saino-Saito et al., 2011). In our subsequent study, DGKζ cytoplasmic translocation is shown to be recapitulated in acute hippocampal slices exposed to oxygen–glucose deprivation (OGD), in which N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx triggers DGKζ cytoplasmic translocation (Suzuki et al., 2012).

Reportedly, cardiac-specific overexpression of DGKζ exerts beneficial effects on a pressure-overloaded or infarcted heart and increases the survival rate compared with that of wild-type mice (Niizeki et al., 2007; Niizeki et al., 2008). In addition, DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity than wild-type neurons (Okada et al., 2012). Together, these results suggest that DGKζ is implicated in a yet undetermined protective role in cellular pathophysiology in cells of various kinds. Nevertheless, it remains unclear how DGKζ nucleocytoplasmic translocation is involved in stress response mechanisms.

In this study, we examined a pathophysiological link between DGKζ cytoplasmic translocation and p53-mediated cytotoxicity after doxorubicin (DOX)-induced DNA damage. For this purpose, using cellular and organismal models, we investigated the effect exerted by DGKζ on p53, i.e. how increased cytoplasmic pool of DGKζ and attenuated nuclear DGKζ affect p53 function after apoptotic stimuli. Overexpression of wild-type DGKζ suppresses p53 induction and reduces apoptosis after DOX-induced DNA damage. Importantly, p53 suppression and the anti-apoptotic effect after DNA damage are more pronounced by an NLS-deleted mutant DGKζΔNLS (cytoplasmic) than by wild-type DGKζ (nuclear predominant). At the organismal level, p53 protein levels are upregulated after kainate-induced seizure as well as under normal unstressed conditions in DGKζ-deficient brain. These findings suggest that (1) DGKζ serves as a sentinel to control p53 function under normal and stressed conditions and (2) its cytoplasmic translocation is a protective stress response and attenuates p53-mediated cytotoxicity under stress conditions. This study provides a novel molecular basis for the regulation of p53-mediated apoptotic pathway.

DGKζ interacts with p53 both in vitro and in vivo

We first examined whether DGKζ physically interacts with p53. Results of co-immunoprecipitation experiments showed that DGKζ associates with p53 in co-transfected HEK293 cells. To map the binding region of DGKζ to p53, cells were co-transfected with FLAG-tagged wild-type DGKζ or its various deletion mutants (Fig. 1A) together with GFP-p53. As shown in Fig. 1B, DGKζΔNLS lacking a nuclear localization signal (NLS; lane 3) and DGKζΔC lacking the C-terminus (lane 4) interacted with p53 with approximately the similar affinity as that of the wild-type DGKζ (lane 2). In contrast, the C-terminus (DGKζC1/3, lane 5) and the N-terminus (DGKζ1-280, lane 6) only slightly associated with p53. These results suggest that the catalytic domain of DGKζ plays a major role in the interaction with p53.

Fig. 1.

DGKζ interacts with p53 both in vitro and in vivo. (A) Schematic representation of truncated or deletion mutants of DGKζ used in the experiment. Zn, zinc finger domain; NLS, nuclear localization signal; catalytic, catalytic domain; Ankyrin repeats, ankyrin-like repeats. (B) HeLa cells were co-transfected with each of the FLAG-tagged constructs in A along with GFP-p53. The lysates were mixed with anti-FLAG antibody combined with protein-G–Sepharose beads and incubated for 6 h at 4°C. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG or anti-GFP antibody. IP, immunoprecipitation; IB, immunoblot. *IgG heavy chain. Immunoprecipitated GFP–p53 were quantified by densitometry and normalized to the value for full-length DGKζ (lane 2). Data shown are the means ± s.d. of three separate experiments. (C) HeLa cells were co-transfected with each of the GFP-tagged p53 constructs, including full-length, N-terminus (p53-N, a.a. 1–101), DNA-binding domain (p53-DB, a.a. 102–292) and C-terminus (p53-C, a.a. 293–393), along with FLAG-DGKζ. Cells were treated and analyzed as in B. Immunoprecipitated FLAG-DGKζ was quantified by densitometry and normalized to the value for full-length p53 (lane 2). Data shown are the means ± s.d. of three separate experiments. (D) HeLa cells were transfected with GFP-DGKζ or GFP-DGKζΔNLS. After 24 h incubation, cells were fixed with 4% paraformaldehyde and analyzed under a confocal microscope. Two hundred cells were counted for the subcellular localization of GFP fluorescence, which was classified into nucleus>cytoplasm, nucleus = cytoplasm, nucleus<cytoplasm. The relative ratios are shown as percentages. (E) HeLa cells were treated with doxorubicin (DOX, 0.2 µg/ml, 12 h) or left untreated,. Total cell lysates were immunoprecipitated with anti-DGKζ antibody or normal rabbit IgG, and the immunoprecipitates were subjected to immunoblotting using anti-p53 or anti-DGKζ antibody. A representative result of three repeated experiments is shown.

Fig. 1.

DGKζ interacts with p53 both in vitro and in vivo. (A) Schematic representation of truncated or deletion mutants of DGKζ used in the experiment. Zn, zinc finger domain; NLS, nuclear localization signal; catalytic, catalytic domain; Ankyrin repeats, ankyrin-like repeats. (B) HeLa cells were co-transfected with each of the FLAG-tagged constructs in A along with GFP-p53. The lysates were mixed with anti-FLAG antibody combined with protein-G–Sepharose beads and incubated for 6 h at 4°C. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG or anti-GFP antibody. IP, immunoprecipitation; IB, immunoblot. *IgG heavy chain. Immunoprecipitated GFP–p53 were quantified by densitometry and normalized to the value for full-length DGKζ (lane 2). Data shown are the means ± s.d. of three separate experiments. (C) HeLa cells were co-transfected with each of the GFP-tagged p53 constructs, including full-length, N-terminus (p53-N, a.a. 1–101), DNA-binding domain (p53-DB, a.a. 102–292) and C-terminus (p53-C, a.a. 293–393), along with FLAG-DGKζ. Cells were treated and analyzed as in B. Immunoprecipitated FLAG-DGKζ was quantified by densitometry and normalized to the value for full-length p53 (lane 2). Data shown are the means ± s.d. of three separate experiments. (D) HeLa cells were transfected with GFP-DGKζ or GFP-DGKζΔNLS. After 24 h incubation, cells were fixed with 4% paraformaldehyde and analyzed under a confocal microscope. Two hundred cells were counted for the subcellular localization of GFP fluorescence, which was classified into nucleus>cytoplasm, nucleus = cytoplasm, nucleus<cytoplasm. The relative ratios are shown as percentages. (E) HeLa cells were treated with doxorubicin (DOX, 0.2 µg/ml, 12 h) or left untreated,. Total cell lysates were immunoprecipitated with anti-DGKζ antibody or normal rabbit IgG, and the immunoprecipitates were subjected to immunoblotting using anti-p53 or anti-DGKζ antibody. A representative result of three repeated experiments is shown.

Next, to map the binding region of p53 to DGKζ (Fig. 1C), cells were co-transfected with FLAG-DGKζ and each of the three parts of p53 tagged with GFP, including the N-terminus (p53-N, lane 3), DNA-binding domain (p53-DB, lane 4), and C-terminus (p53-C, lane 5). Results show that DGKζ efficiently interacted with the DNA-binding domain of p53 (lane 4), although it also associated with the N- and C-termini of p53 weakly (lanes 3 and 5). It is particularly interesting that DGKζ binding affinity to the DNA-binding domain of p53 (lane 4) was much higher than that to the full-length p53 (lane 2). These results suggest that the DNA-binding domain is the primary site for p53 binding to DGKζ although the N- and C-termini also affect p53 binding affinity to DGKζ. It is noteworthy that, in terms of the subcellular site for the binding, DGKζΔNLS, which localizes almost exclusively to the cytoplasm, exhibited the similar binding affinity as wild-type DGKζ, which localizes predominantly to the nucleus (Fig. 1D), suggesting that DGKζ interacts with p53 both in the nucleus and cytoplasm.

We further investigated the interaction between p53 and DGKζ at the endogenous level. We performed co-immunoprecipitation assay of HeLa cells using specific antibodies (Fig. 1E). Endogenous p53 was detected in the immunoprecipitates of anti-DGKζ antibody in cells left untreated (lane 3) and treated with DOX (lane 4), and the amount of p53 protein bound to DGKζ in the presence or absence of DOX treatment (DOX +/−) was not changed significantly [relative ratio of p53 binding was calculated as 1.26±0.27 (mean ± s.d.), n = 3, statistically not significant]. We confirmed the specificity of the interaction between DGKζ and p53 by the experiment using siRNA for DGKζ (supplementary material Fig. S9). This result suggests that DGKζ interacts with p53 endogenously, independent of DNA damage.

Cytoplasmic DGKζΔNLS potently suppresses p53 induction and apoptosis in a kinase-independent manner after treatment with DOX

A cellular model of DOX-induced DNA damage was established according to a previous study (Schäfer et al., 1998). We treated HeLa cells with varying doses of DOX and assessed the protein levels of p53 (supplementary material Fig. S1A). Treatment of cells at a dose of 0.2 µg/ml of DOX gradually increased the levels of p53 protein to 24 h. During the course of DOX treatment at this concentration, DGKζ showed no marked change in the protein levels (supplementary material Fig. S1B). For the following experiments, we used HeLa cells treated with 0.2 µg/ml DOX as a cellular model of DNA damage unless otherwise noted.

To explore the functional relation between DGKζ cytoplasmic translocation and apoptotic pathway involving p53, we tested whether wild-type DGKζ and DGKζΔNLS affect the protein levels of p53 after DNA damage. HeLa cells were transfected with empty vector, FLAG-DGKζ or FLAG-DGKζΔNLS. After treatment with DOX for 24 h, or being left untreated, cells were examined using immunoblot analysis (Fig. 2A). In response to DOX-induced DNA damage, p53 protein was induced at about fivefold in control cells transfected with vector alone. However, in cells transfected with wild-type DGKζ or cytoplasmic DGKζΔNLS p53 induction was at 3.6-fold or 1.7-fold, respectively (Fig. 2B). We also used other cell lines, human neuroblastoma SHSY5Y cells (supplementary material Fig. S2) and osteosarcoma U2OS cells (supplementary material Fig. S7A), with DOX, and obtained similar results. Furthermore, we confirmed that cytoplasmic DGKζΔNLS suppressed p53 induction dose-dependently (supplementary material Fig. S3). Concomitantly, overexpression of DGKζ suppressed an apoptotic marker (anti-cleaved PARP antibody). Notably, cytoplasmic DGKζΔNLS did so to a greater extent compared with wild-type DGKζ (Fig. 2A, lane 6). Consistent with the attenuation of 53 induction, DGKζΔNLS suppressed p53-inducible gene products, p21 and Bax. It should be mentioned that DGKζΔNLS-mediated attenuation of cleaved PARP levels was abrogated by p53 knockdown (supplementary material Fig. S6A, lanes 1, 2, 3 versus lanes 4, 5, 6), suggesting that DGKζΔNLS regulates cleaved PARP levels through p53. However, wild-type DGKζ and cytoplasmic DGKζΔNLS exerted no apparent suppressive effect on the induction of E2F1 (Fig. 2A, lanes 4 and 6), another transcription factor that is induced to accumulate in response to DNA damage and engenders apoptosis (supplementary material Fig. S1B) (Lin et al., 2001; Wu and Levine, 1994). These results suggest that wild-type DGKζ, and more prominently, cytoplasmic DGKζΔNLS, suppress p53 induction, but that they have no effect on E2F1 induction.

Fig. 2.

Cytoplasmic DGKζΔNLS potently suppresses p53 induction in a kinase-independent manner. (A) HeLa cells were transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS, and treated with DOX (0.2 µg/ml, 12 h) or left untreated. Total cell lysates (20 µg) were analyzed by immunoblotting using antibodies against p53 (DO-1), cleaved PARP (#5625), Mdm2 (Ab-1), p21 (OP64), Bax (sc-526), E2F1 (KH20 and KH95) and FLAG. Blots were reprobed with β-actin antibody to confirm equal loading of the samples. Immunoblot signals were quantified by densitometry and normalized to the value obtained in cells without treatment (for p53) or to the one obtained in cells transfected with vector (for cleaved PARP). (B) Relative ratio of p53 induction (DOX +/−) in A. Data shown are the means ± s.d. of five separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (C) HeLa cells were transfected with either control vector, FLAG-DGKζΔNLS or FLAG-DGKζΔNLS-KD (kinase dead mutant of DGKζΔNLS) and treated with or without DOX (0.2 µg/ml, 12 h). Cell lysates (20 µg) were analyzed as in A. A representative result of three repeated experiments is shown.

Fig. 2.

Cytoplasmic DGKζΔNLS potently suppresses p53 induction in a kinase-independent manner. (A) HeLa cells were transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS, and treated with DOX (0.2 µg/ml, 12 h) or left untreated. Total cell lysates (20 µg) were analyzed by immunoblotting using antibodies against p53 (DO-1), cleaved PARP (#5625), Mdm2 (Ab-1), p21 (OP64), Bax (sc-526), E2F1 (KH20 and KH95) and FLAG. Blots were reprobed with β-actin antibody to confirm equal loading of the samples. Immunoblot signals were quantified by densitometry and normalized to the value obtained in cells without treatment (for p53) or to the one obtained in cells transfected with vector (for cleaved PARP). (B) Relative ratio of p53 induction (DOX +/−) in A. Data shown are the means ± s.d. of five separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (C) HeLa cells were transfected with either control vector, FLAG-DGKζΔNLS or FLAG-DGKζΔNLS-KD (kinase dead mutant of DGKζΔNLS) and treated with or without DOX (0.2 µg/ml, 12 h). Cell lysates (20 µg) were analyzed as in A. A representative result of three repeated experiments is shown.

We next examined whether the DGK activity is necessary for the suppressive effect on p53 induction (Fig. 2C). HeLa cells transfected with either empty vector, FLAG-DGKζΔNLS, or its kinase-dead mutant FLAG-DGKζΔNLS-KD, were treated with DOX. The p53 induction was suppressed in cells transfected with FLAG-DGKζΔNLS-KD (lane 6), seemingly to the same degree as FLAG-DGKζΔNLS (lane 4), suggesting that DGKζΔNLS suppresses p53 induction independently of its kinase activity.

As a next step, we performed Crystal Violet and water soluble tetrazolium salt (WST-1) assays to examine cell viability under the same conditions as those described above. Crystal violet assay showed clearly that cells transfected with DGKζΔNLS were more viable than those transfected with control vector (Fig. 3A). In the presence of siRNA for p53, cells transfected with DGKζΔNLS showed cell viability similar to those transfected with control vector and FLAG-DGKζ (supplementary material Fig. S6B), suggesting that the effect exerted by DGKζΔNLS after DNA damage is p53-dependent. WST-1 assay revealed quantitatively that cell viability after DNA damage was increased in cells transfected with DGKζ and more prominently with DGKζΔNLS (Fig. 3B). As another control, we also used DGKι, which belongs to the same subclass as DGKζ but which localizes to the cytoplasm (Ito et al., 2004). DGKι had no apparent effect on cell viability after DNA damage (Fig. 3C). Collectively, these results suggest that wild-type DGKζ, and more potently, cytoplasmic DGKζΔNLS, have anti-apoptotic effects and increase cell viability after DNA damage through the suppression of p53 induction.

Fig. 3.

Cytoplasmic DGKζΔNLS attenuates apoptosis after DOX treatment. (A,B) HeLa cells were transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS and treated with or without DOX (0.2 µg/ml, 24 h), and analyzed for cell viability by Crystal Violet assay (A) and WST-1 assay (B), as described in Materials and Methods. Data shown are the means ± s.d. of three separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (C) HeLa cells were transfected with control vector or GFP-DGKι and analyzed for cell viability with the WST-1 assay. Note that DGKι does not affect cell viability after DOX treatment. (D) HeLa cells were transfected with siRNA control (siCont) or siRNA against human DGKζ (siDGKζ-1). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, and then analysed by immunoblotting (20 µg) using antibodies against cleaved PARP (Ab-1), p53 (DO-1) and DGKζ. β-actin was used as a control. A representative result of three repeated experiments is shown.

Fig. 3.

Cytoplasmic DGKζΔNLS attenuates apoptosis after DOX treatment. (A,B) HeLa cells were transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS and treated with or without DOX (0.2 µg/ml, 24 h), and analyzed for cell viability by Crystal Violet assay (A) and WST-1 assay (B), as described in Materials and Methods. Data shown are the means ± s.d. of three separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (C) HeLa cells were transfected with control vector or GFP-DGKι and analyzed for cell viability with the WST-1 assay. Note that DGKι does not affect cell viability after DOX treatment. (D) HeLa cells were transfected with siRNA control (siCont) or siRNA against human DGKζ (siDGKζ-1). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, and then analysed by immunoblotting (20 µg) using antibodies against cleaved PARP (Ab-1), p53 (DO-1) and DGKζ. β-actin was used as a control. A representative result of three repeated experiments is shown.

To elucidate the physiological importance of DGKζ, we examined the functional consequences of siRNA-mediated knockdown of endogenous DGKζ. HeLa cells, which express both DGKζ and wild-type p53 proteins, were transfected with control siRNA (siControl) or siRNA for DGKζ (siDGKζ). As shown in Fig. 3D, siDGKζ attenuated endogenous DGKζ whereas the levels of control β-actin protein remained the same. Importantly, knockdown of DGKζ strongly augmented p53 induction after DOX-induced DNA damage (lane 4). In addition, an apoptotic marker (cleaved PARP) was increased significantly in cells treated with siDGKζ. In the presence of siRNA for p53, cleaved PARP levels were not significantly changed in cells transfected with siControl and siDGKζ/sip53 (Fig. 3E, lane 3 versus lane 4), suggesting that DGKζ regulates cleaved PARP levels through p53. These results suggest that knockdown of endogenous DGKζ significantly enhances p53 induction and p53-dependent apoptotic pathway after DNA damage.

DGKζΔNLS induces cytoplasmic localization of p53 and enhances its degradation through the ubiquitin–proteasome system in the cytoplasm

Nuclear export of p53 has been reportedly involved in its degradation (Freedman and Levine, 1998). Therefore, we investigated the effect of cytoplasmic DGKζΔNLS on p53 subcellular localization in transfected HeLa cells. HeLa cells were co-transfected with GFP-p53 and either FLAG-DGKζ or FLAG-DGKζΔNLS (Fig. 4A). In control cells co-transfected with control vector, GFP–p53 localized mainly to the nucleus (top panels). In cells co-transfected with FLAG-DGKζ, both FLAG-DGKζ and GFP-p53 mostly localized to the nucleus (middle panels, arrows). However, in cells co-transfected with DGKζΔNLS, which mostly localizes to the cytoplasm, a significant part of p53 was detected in the cytoplasm and colocalized with DGKζΔNLS (lower panels, asterisks). In our observation, most of the cells transfected with DGKζΔNLS (about 80%) showed a variable degree of cytoplasmic localization of p53. These results suggest that cytoplasmic DGKζΔNLS induces cytoplasmic localization of ectopic p53.

Fig. 4.

Cytoplasmic DGKζΔNLS induces cytoplasmic localization of p53 and enhances its degradation through the ubiquitin–proteasome system in the cytoplasm. (A) HeLa cells were co-transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS along with GFP-p53. After 24 h incubation, cells were fixed with 4% paraformaldehyde and stained with anti-FLAG antibody followed by anti-mouse IgG-conjugated Alexa Fluor 546 (red). They were analyzed for double fluorescence under a confocal microscopy. Arrows indicate nuclear localization of FLAG–DGKζ and GFP–p53. In co-transfected cells with DGKζΔNLS, GFP–p53 was partially induced to localize to the cytoplasm (asterisks). (B) HeLa cells were transfected with either vector, FLAG-DGKζ or FLAG-DGKζΔNLS together with GFP–p53. After 12 h incubation with DOX (0.2 µg/ml) and MG132 (5 µM), the lysates were mixed with anti-GFP antibody and then with protein-G-conjugated Sepharose beads, and incubated for 6 h at 4°C. The immunoprecipitates were analyzed by immunoblotting using anti-ubiquitin (U5379) or anti-GFP antibody. β-actin was used as a control. (C) HeLa cells were transfected with either control vector, FLAG-DGKζ, FLAG-DGKζΔNLS, siRNA control (siCont) or siRNA against human DGKζ (siDGKζ-1). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, followed by treatment with leptomycin B (LMB, 5 ng/ml, 12 h) or MG132 (5 µM, 8 h). Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against p53 (DO-1) and FLAG or anti-DGKζ. (D) HeLa cells were transfected with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-2). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, and subjected to immunoblotting (20 µg) using antibodies against p53 (DO-1), Mdm2 (Ab-1) and DGKζ. Note that DGKζ knockdown with siDGKζ-2 augments p53 induction after DOX treatment, similarly to that with siDGKζ-1 (Fig. 3D). (E) HeLa cells were co-transfected with siRNA control (siCont) or siRNA against human Mdm2 (siMdm2) along with GFP vector or GFP-DGKζΔNLS. After 48 h incubation, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated, and cell lysates (20 µg) were subjected to immunoblotting using antibodies against p53 (DO-1), Mdm2 (Ab-1) and GFP. A representative result of three repeated experiments is shown.

Fig. 4.

Cytoplasmic DGKζΔNLS induces cytoplasmic localization of p53 and enhances its degradation through the ubiquitin–proteasome system in the cytoplasm. (A) HeLa cells were co-transfected with either control vector, FLAG-DGKζ or FLAG-DGKζΔNLS along with GFP-p53. After 24 h incubation, cells were fixed with 4% paraformaldehyde and stained with anti-FLAG antibody followed by anti-mouse IgG-conjugated Alexa Fluor 546 (red). They were analyzed for double fluorescence under a confocal microscopy. Arrows indicate nuclear localization of FLAG–DGKζ and GFP–p53. In co-transfected cells with DGKζΔNLS, GFP–p53 was partially induced to localize to the cytoplasm (asterisks). (B) HeLa cells were transfected with either vector, FLAG-DGKζ or FLAG-DGKζΔNLS together with GFP–p53. After 12 h incubation with DOX (0.2 µg/ml) and MG132 (5 µM), the lysates were mixed with anti-GFP antibody and then with protein-G-conjugated Sepharose beads, and incubated for 6 h at 4°C. The immunoprecipitates were analyzed by immunoblotting using anti-ubiquitin (U5379) or anti-GFP antibody. β-actin was used as a control. (C) HeLa cells were transfected with either control vector, FLAG-DGKζ, FLAG-DGKζΔNLS, siRNA control (siCont) or siRNA against human DGKζ (siDGKζ-1). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, followed by treatment with leptomycin B (LMB, 5 ng/ml, 12 h) or MG132 (5 µM, 8 h). Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against p53 (DO-1) and FLAG or anti-DGKζ. (D) HeLa cells were transfected with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-2). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated, and subjected to immunoblotting (20 µg) using antibodies against p53 (DO-1), Mdm2 (Ab-1) and DGKζ. Note that DGKζ knockdown with siDGKζ-2 augments p53 induction after DOX treatment, similarly to that with siDGKζ-1 (Fig. 3D). (E) HeLa cells were co-transfected with siRNA control (siCont) or siRNA against human Mdm2 (siMdm2) along with GFP vector or GFP-DGKζΔNLS. After 48 h incubation, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated, and cell lysates (20 µg) were subjected to immunoblotting using antibodies against p53 (DO-1), Mdm2 (Ab-1) and GFP. A representative result of three repeated experiments is shown.

To examine how p53 induction is attenuated efficiently by cytoplasmic DGKζΔNLS, we first performed RT-PCR analysis. HeLa cells were transfected with empty vector or DGKζΔNLS in the absence or presence of DOX. Levels of p53 mRNA showed no apparent changes before and after DNA damage (supplementary material Fig. S4), suggesting that p53 is not regulated at the transcriptional level. The tight control of cellular p53 levels is achieved primarily through ubiquitin–proteasomal degradation (Brooks and Gu, 2006; Kruse and Gu, 2009; Michael and Oren, 2003). To examine whether the ubiquitin–proteasome system (UPS) is involved in the attenuated p53 induction by DGKζ and DGKζΔNLS after DNA damage, we used a proteasome inhibitor MG132 to stabilize ubiquitinated proteins. HeLa cells were co-transfected with GFP-p53 and either empty vector, FLAG-DGKζ or FLAG-DGKζΔNLS in the presence of 5 µM MG132 for 8 h (Fig. 4B). After treatment with DOX, highly ubiquitinated species of p53 were detected abundantly in cells that had been co-transfected with wild-type DGKζ and more prominently with DGKζΔNLS compared with control vector. Similar results were obtained in U2OS cells (supplementary material Fig. S7B). Therefore, p53 was presumably degraded through the UPS in the cytoplasm. To verify this inference, we treated the transfected cells with leptomycin B (LMB) to block nuclear export of p53 or with proteasome inhibitor MG132. As shown in Fig. 4C, LMB treatment abolished the suppression of p53 induction in cells transfected with DGKζΔNLS (lane 14 versus lane 16). A similar result was obtained in the presence of MG132 (lane 14 versus lane 15). When we knocked down DGKζ by its siRNA, p53 levels were not attenuated in the presence of DOX treatment (lane 18 versus lane 22) and LMB or MG132 treatment had no significant effect on p53 levels (lane 22 versus lanes 23 and 24). Collectively, these results suggest that p53 associated with cytoplasmic DGKζΔNLS is degraded through cytoplasmic UPS.

E3 ubiquitin ligase Mdm2 plays a critical role in maintaining p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteasome (Manfredi, 2010; Marine and Lozano, 2010). Therefore, we next asked whether Mdm2 is involved in p53 degradation induced by cytoplasmic DGKζΔNLS. In the presence of DOX treatment, Mdm2 levels were reduced in cells transfected with control vector (Fig. 2A, lane 1 versus lane 2) whereas its levels were not attenuated and remained high in cells transfected with DGKζΔNLS (Fig. 2A, lane 5 versus lane 6). When we knocked down DGKζ by its siRNA, Mdm2 was also downregulated and p53 levels were increased in the presence of DOX treatment (Fig. 4D, lane 3 versus lane 4). In addition, when Mdm2 was knocked down by its siRNA, increased p53 levels in the presence of DOX treatment was not attenuated by DGKζΔNLS (Fig. 4E, lane 6 versus lane 8), suggesting that cytoplasmic DGKζ facilitates p53 degradation through Mdm2 after DOX treatment.

Overexpression of cytoplasmic DGKζΔNLS has no effect on apoptosis in p53-deficient Saos2 cells after DNA damage

Having shown that cytoplasmic DGKζΔNLS inhibits apoptosis by attenuating p53 induction after DNA damage, we asked further if the anti-apoptotic effect of cytoplasmic DGKζΔNLS is mediated by attenuation of p53 induction using p53-deficient Saos2 cells. Saos2 cells were transfected with either empty vector or FLAG-DGKζΔNLS. After treatment with DOX for 24 h or being left untreated, cells were subjected to immunoblot analysis. As shown in Fig. 5, the intensity of the apoptotic marker (cleaved PARP) after DOX treatment was not significantly different among cells transfected with the control vector (lane 2) or DGKζΔNLS (lane 4). Importantly, in an add-back experiment in which GFP-p53 was reintroduced, cytoplasmic DGKζΔNLS resumed downregulation of the apoptotic marker after DOX treatment (lane 6 versus lane 8). Collectively, these results confirm that the protective effect of cytoplasmic DGKζΔNLS after DNA damage is dependent on p53.

Fig. 5.

Cytoplasmic DGKζΔNLS has no effect on DOX-induced apoptosis in p53-deficient Saos2 cells. Saos2 cells (p53-null) were transfected with either GFP vector (lanes 1 and 2) or GFP-DGKζΔNLS (lanes 3 and 4). In an add-back experiment, Saos2 cells were co-transfected with FLAG-p53 along with either GFP vector (lanes 5 and 6) or GFP-DGKζΔNLS (lanes 7 and 8). After 48 h, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), FLAG GFP and β-actin. Immunoblot signals were quantified by densitometry and normalized to the value obtained in control cells transfected with GFP vector. A representative result of three repeated experiments is shown.

Fig. 5.

Cytoplasmic DGKζΔNLS has no effect on DOX-induced apoptosis in p53-deficient Saos2 cells. Saos2 cells (p53-null) were transfected with either GFP vector (lanes 1 and 2) or GFP-DGKζΔNLS (lanes 3 and 4). In an add-back experiment, Saos2 cells were co-transfected with FLAG-p53 along with either GFP vector (lanes 5 and 6) or GFP-DGKζΔNLS (lanes 7 and 8). After 48 h, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), FLAG GFP and β-actin. Immunoblot signals were quantified by densitometry and normalized to the value obtained in control cells transfected with GFP vector. A representative result of three repeated experiments is shown.

Cytoplasmic DGKζΔNLS effect is recapitulated by co-expression of wild-type DGKζ and its binding partners nucleosome assembly protein 1Ls (NAP1Ls), which translocate DGKζ to the cytoplasm

We showed that cytoplasmic DGKζΔNLS suppresses p53 induction and exerts an anti-apoptotic effect after DNA damage. We next examined whether wild-type DGKζ might exert the same effect when it translocates from the nucleus to the cytoplasm. Recently we reported that novel DGKζ binding partners NAP1L1 and NAP1L4 induce cytoplasmic translocation of DGKζ in HEK293 cells (Okada et al., 2011). Similarly, in HeLa cells co-transfected with either GFP-NAP1L1 or GFP-NAP1L4 together with wild-type DGKζ tagged with mCherry, NAP1Ls induced cytoplasmic translocation of mCherry-DGKζ and endogenous p53 (Fig. 6A, asterisks; the ratios of cells showing cytoplasmic dominant DGKζ and cytoplasmic staining of p53 were roughly estimated as 25%, 90%, and 80% in cells cotransfected with vector, NAP1L1, and NAP1L4, respectively). Notably, transfection of NAP1L1 or NAP1L4 alone in HeLa cells suppressed endogenous p53 induction and the apoptotic marker after DNA damage (Fig. 6B, lanes 4 and 6). In addition, transfection of NAP1L1 or NAP1L4 alone exerted a protective effect after DOX-induced cytotoxicity as assessed using WST-1 assay (Fig. 6C), which is consistent with our previous study (Okada et al., 2011). In this setting, p53 suppression effect by NAP1L1 and NAP1L4 was abrogated by knockdown of DGKζ (Fig. 6D, lanes 8 and 12), suggesting that an anti-apoptotic effect is mediated through DGKζ that is translocated from the nucleus to the cytoplasm, but not NAP1Ls themselves. Together, these results indicate that the cytoplasmic DGKζΔNLS effect is recapitulated by cytoplasmic translocation of wild-type DGKζ.

Fig. 6.

Co-expression of wild-type DGKζ and its binding partners nucleosome assembly protein 1Ls (NAP1Ls) recapitulates cytoplasmic DGKζΔNLS effect. (A) HeLa cells were co-transfected with either control vector, GFP-NAP1L1 or GFP-NAP1L4 along with mCherry-DGKζ. After 24 h incubation, cells were fixed and stained with anti-p53 antibody (DO-1). p53 is localized to the nucleus in cells co-transfected with control vector and mCherry-DGKζ (upper panels). However, in cells co-transfected with GFP-NAP1L1 (middle panels) or GFP-NAP1L4 (lower panels), a significant part of p53 is localized to the cytoplasm (asterisks) along with mCherry-DGKζ, which is mostly translocated to the cytoplasm (asterisks). (B) HeLa cells were transfected with either control vector, GFP-NAP1L1 or GFP-NAP1L4 and treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), p53 (DO-1), DGKζ and GFP. β-actin was used as a control. Immunoblot signals were quantified by densitometry and normalized to the value obtained in control cells transfected with GFP vector. (C) HeLa cells in B were analyzed for cell viability by WST-1 assay. Data shown are the means ± s.d. of three separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (D) HeLa cells were co-transfected with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-1) along with either GFP vector (left panels), GFP-NAP1L1 (middle panels) or GFP-NAP1L4 (right panels). After 48 h, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), p53 (DO-1), DGKζ and GFP. A representative result of three repeated experiments is shown.

Fig. 6.

Co-expression of wild-type DGKζ and its binding partners nucleosome assembly protein 1Ls (NAP1Ls) recapitulates cytoplasmic DGKζΔNLS effect. (A) HeLa cells were co-transfected with either control vector, GFP-NAP1L1 or GFP-NAP1L4 along with mCherry-DGKζ. After 24 h incubation, cells were fixed and stained with anti-p53 antibody (DO-1). p53 is localized to the nucleus in cells co-transfected with control vector and mCherry-DGKζ (upper panels). However, in cells co-transfected with GFP-NAP1L1 (middle panels) or GFP-NAP1L4 (lower panels), a significant part of p53 is localized to the cytoplasm (asterisks) along with mCherry-DGKζ, which is mostly translocated to the cytoplasm (asterisks). (B) HeLa cells were transfected with either control vector, GFP-NAP1L1 or GFP-NAP1L4 and treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), p53 (DO-1), DGKζ and GFP. β-actin was used as a control. Immunoblot signals were quantified by densitometry and normalized to the value obtained in control cells transfected with GFP vector. (C) HeLa cells in B were analyzed for cell viability by WST-1 assay. Data shown are the means ± s.d. of three separate experiments. Asterisk indicates significance, *P<0.01 (Student's t-test). (D) HeLa cells were co-transfected with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-1) along with either GFP vector (left panels), GFP-NAP1L1 (middle panels) or GFP-NAP1L4 (right panels). After 48 h, cells were treated with DOX (0.2 µg/ml, 24 h) or left untreated. Cell lysates (20 µg) were analyzed by immunoblotting using antibodies against cleaved PARP (#5625), p53 (DO-1), DGKζ and GFP. A representative result of three repeated experiments is shown.

p53 induction is enhanced in DGKζ-deficient brain under normal conditions and after kainate-induced seizures

Previous reports have described that p53 induction is associated with excitotoxicity after systemic administration of kainate, a potent excitotoxin that produces seizures followed by a defined pattern of neuronal cell loss (Araki et al., 2004; Sakhi et al., 1994). In addition, mice deficient in p53 exhibit almost complete protection from brain injury induced by kainate seizures (Morrison et al., 1996; Xiang et al., 1996). In this regard, we previously reported that DGKζ translocates from the nucleus to the cytoplasm in hippocampal neurons in cellular (Okada et al., 2012) and animal models (Saino-Saito et al., 2011) of kainate exposure. The present study revealed that overexpression of cytoplasmic DGKζΔNLS suppresses p53 induction and that knockdown of DGKζ conversely enhances p53 induction in a cellular model of DNA damage. Therefore, we investigated whether DGKζ regulates p53 induction at the organismal level using an animal model of kainate-induced seizures on DGKζ-deficient mice. Mice injected systemically with kainate (25 mg/kg, i.p.) suffered from compiled tonic seizures of stage 5, during which about 90% of them survived for 24 h. Consistent with the data obtained in a cellular model, p53 levels were strongly upregulated in DGKζ-deficient brain after kainate-induced seizures compared with those in wild-type brain (Fig. 7, lane 2 versus lanes 6, 7, 8). Notably, p53 levels were also upregulated in DGKζ-deficient brain under normal conditions (lane 1 versus lanes 3, 4, 5).

Fig. 7.

p53 is upregulated in DGKζ-deficient brain under normal conditions and after kainate-induced seizures. Wild-type (lane 2) and DGKζ-deficient mice (lanes 6, 7, 8) were injected intraperitoneally with kainate (KA, 25 mg/kg) and observed for seizure activity. Saline was used as a negative control (lanes 1, 3, 4, 5). After 24 h, whole brains were removed and homogenized in lysis buffer. Total lysates (50 µg) were subjected to immunoblot analysis using antibodies against DGKζ and p53 (Ab-4). β-actin was used as a control. Immunoblot signals were quantified by densitometry and normalized to the value obtained from brain samples from wild-type mice without KA treatment.

Fig. 7.

p53 is upregulated in DGKζ-deficient brain under normal conditions and after kainate-induced seizures. Wild-type (lane 2) and DGKζ-deficient mice (lanes 6, 7, 8) were injected intraperitoneally with kainate (KA, 25 mg/kg) and observed for seizure activity. Saline was used as a negative control (lanes 1, 3, 4, 5). After 24 h, whole brains were removed and homogenized in lysis buffer. Total lysates (50 µg) were subjected to immunoblot analysis using antibodies against DGKζ and p53 (Ab-4). β-actin was used as a control. Immunoblot signals were quantified by densitometry and normalized to the value obtained from brain samples from wild-type mice without KA treatment.

Knockdown of DGKζ attenuates p53 transactivation activity

p53 is well known to accumulate in the nucleus and to act as a nuclear transcription factor that transactivates genes involved in cell cycle regulation, apoptosis, and numerous other processes (Chao et al., 2000; Kruse and Gu, 2009). Because DGKζ interacted efficiently with the DNA-binding domain of p53 (Fig. 1C), we next investigated whether downregulation of nuclear DGKζ affects the p53 transcriptional activity. To address this issue, we first performed p53 reporter assay (Fig. 8A) under the same experimental conditions as those shown in Fig. 4D, i.e. in HeLa cells co-transfected with either siControl or siDGKζ along with p53 reporter (pp53-Luc) in the DNA damage-dependent p53 response. p53 transcriptional activity was repressed significantly, but not completely, in HeLa cells transfected with siDGKζ compared with those transfected with siControl in the absence of DOX treatment, i.e. without DNA damage (Fig. 8A, lane 1 versus lane 3). Surprisingly, p53 transcriptional activity was also repressed in HeLa cells transfected with siDGKζ in the presence of DOX treatment, i.e. after DNA damage, even though p53 was potently induced (Fig. 8A, lane 2 versus lane 4). We repeated the experiment using p53-deficient Saos2 cells under ectopic p53 expression. We transfected Saos2 cells with either siControl or siDGKζ along with pp53-Luc and FLAG-p53, and confirmed that p53 transcriptional activity was attenuated in DGKζ-knocked down cells upon ectopic p53 expression (supplementary material Fig. S5). We also performed an additional reporter assay using AP-1 reporter (pAP1-Luc) to examine whether downregulation of DGKζ affects other transcription factors. Results show that siDGKζ exerted no significant effect on AP-1 transcriptional activity (supplementary material Fig. S8), suggesting that knockdown of DGKζ attenuates specifically, if not exclusively, p53 transactivation activity.

Fig. 8.

p53 reporter assay. (A) Knockdown of DGKζ attenuates p53 transactivation activity. HeLa cells were co-transfected with pp53-Luc and pRL-null (internal control) reporter constructs along with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-2). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated. Cell lysates (20 µg) were analyzed for p53 transcriptional activity and by immunoblotting using antibodies against DGKζ, p53 (DO-1), Bax (sc-526), p21 (OP64) and β-actin. The bar graph represents the normalized luciferase reporter activity of five independent experiments; shown as the means ± s.e.m. Asterisks indicate significance, *P<0.01 (Student's t-test). A representative result of three repeated experiments is shown. (B) A model depicting the effect of DGKζ nucleocytoplasmic translocation on p53 under stress conditions. Under stress conditions, nuclear DGKζ is required for p53 transactivation activity. Decreased levels of nuclear DGKζ leads to suppression of the transcriptional activity of p53. Once translocated to the cytoplasm, e.g. through binding with the NAP proteins, cytoplasmic DGKζ acts as a scaffolding protein to promote p53 degradation through a Mdm2-dependent ubiquitin-proteasome pathway, resulting in attenuation of p53-mediated cytotoxicity.

Fig. 8.

p53 reporter assay. (A) Knockdown of DGKζ attenuates p53 transactivation activity. HeLa cells were co-transfected with pp53-Luc and pRL-null (internal control) reporter constructs along with control siRNA (siCont) or human DGKζ-siRNA (siDGKζ-2). After 48 h, cells were treated with DOX (0.2 µg/ml, 12 h) or left untreated. Cell lysates (20 µg) were analyzed for p53 transcriptional activity and by immunoblotting using antibodies against DGKζ, p53 (DO-1), Bax (sc-526), p21 (OP64) and β-actin. The bar graph represents the normalized luciferase reporter activity of five independent experiments; shown as the means ± s.e.m. Asterisks indicate significance, *P<0.01 (Student's t-test). A representative result of three repeated experiments is shown. (B) A model depicting the effect of DGKζ nucleocytoplasmic translocation on p53 under stress conditions. Under stress conditions, nuclear DGKζ is required for p53 transactivation activity. Decreased levels of nuclear DGKζ leads to suppression of the transcriptional activity of p53. Once translocated to the cytoplasm, e.g. through binding with the NAP proteins, cytoplasmic DGKζ acts as a scaffolding protein to promote p53 degradation through a Mdm2-dependent ubiquitin-proteasome pathway, resulting in attenuation of p53-mediated cytotoxicity.

We next examined whether this regulation of p53 transactivation activity by DGKζ is exerted on pro-arrest or pro-apoptotic p53 genes. Immunoblot analysis of HeLa cell experiment described (Fig. 8A) revealed that DGKζ knockdown reduces p53-dependent induction of both pro-arrest p53 target p21 and pro-apoptotic target Bax (lane 2 versus lane 4). Collectively, these results suggest that attenuation of nuclear DGKζ might suppress p53-dependent genes.

We previously demonstrated that DGKζ nucleocytoplasmic translocation is induced in hippocampal neurons in animal models of global ischemia (Ali et al., 2004) and kainate-induced seizures (Saino-Saito et al., 2011), in acute hippocampal slices exposed to oxygen-glucose deprivation (OGD) (Suzuki et al., 2012), and in a cellular model of kainate excitotoxicity using primary cultured neurons (Okada et al., 2012). From these observations, we predicted that DGKζ would be involved in stress responses. However, how is DGKζ involved in this process? Previous reports have described that nuclear DGKζ negatively regulates cell cycle progression. Overexpression of DGKζ causes accumulation of cells in G0/G1 phase of the cell cycle (Topham et al., 1998). Both DGK activity and a functional NLS are necessary to block the cell cycle at G0/G1, suggesting that the cell cycle blockage results from DGKζ-mediated metabolism of nuclear DG. In addition, DGKζ interacts with the retinoblastoma protein (pRb) depending on its phosphorylation status (Los et al., 2006), and overexpression of DGKζ is accompanied by decreased levels of pRb phosphorylated on Ser807/811 (Evangelisti et al., 2007). Furthermore, in C2C12 mouse myoblasts, nuclear DGKζ downregulates the expression of cyclin D through upregulation of TIS21/BTG2/PC3, a transcriptional regulator of cyclin D1 with a strong anti-proliferative function (Evangelisti et al., 2009). Therefore, nuclear DGKζ might participate in an anti-proliferative safeguard mechanism to suppress neuronal cell cycle progression. Relaxation of this vigilance puts post-mitotic neurons at risk of unscheduled cell cycle progression, which engenders apoptosis. In accord with this hypothesis, DGKζ-deficient neurons are shown to be more vulnerable to excitotoxicity than wild-type ones because of aberrant cell cycle re-entry (Okada et al., 2012). However, much remains to be elucidated about functional implications of DGKζ in the cytoplasm.

In the present study, we investigated functional links between DGKζ nucleocytoplasmic translocation and p53 that serves as the hub of numerous apoptotic signaling pathways under various stress conditions including excitotoxicity. Here we show that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity under stress conditions. Results indicate that cytoplasmic DGKζ anchors p53 and facilitates its degradation through the UPS in the cytoplasm. WST-1 assay confirms its cytoprotective effect against DNA damage. It is noteworthy that all events mediated by the NLS-deleted mutant DGKζΔNLS, which resides in the cytoplasm, are recapitulated by co-expression of nuclear-resident wild-type DGKζ and its binding partners NAP1Ls, which are shown to translocate wild-type DGKζ to the cytoplasm by attenuating its association with importins (Okada et al., 2011). Because knockdown of DGKζ abolishes p53 suppression and cytoprotective effects of NAP1Ls, these effects are thought to be exerted by DGKζ cytoplasmic translocation. NAP is a highly conserved histone chaperone protein that is involved in the dynamic regulation of the H2A-H2B histone heterodimer (Zlatanova et al., 2007). Furthermore, NAP1 is shown to interact functionally with the transcriptional activator E2 together with p300, the transcriptional co-activator possessing histone acetyltransferase activity, suggesting a direct contribution of NAP1 in the regulation of the transcriptional machinery (Rehtanz et al., 2004). In the present study, we provide evidence that an anti-apoptotic effect of NAP1Ls is mediated through DGKζ cytoplasmic translocation, which proposes a potential novel role of NAP under stress conditions. It would be warranted to dissect the molecular basis of the interaction between DGKζ and NAP1Ls under stress conditions.

How does DGKζ attenuate p53 induction? Our results show that cytoplasmic DGKζ stabilizes Mdm2 levels whereas DGKζ knockdown attenuates its levels after DOX treatment. In addition, knockdown of Mdm2 abrogates cytoplasmic DGKζ-induced p53 attenuation. These results suggest that DGKζ attenuates p53 induction through Mdm2 in the cytoplasm under stress conditions. Mdm2 is a very short-lived protein, whose rapid degradation results from ubiquitin-dependent proteolysis (Chang et al., 1998). In addition to ubiquitinating p53, Mdm2 can catalyze its own ubiquitination (Fang et al., 2000; Honda and Yasuda, 2000), although a recent study suggests that Mdm2 stability is controlled by another E3 ubiquitin ligase in vivo, such as histone acetyltransferase PCAF (Linares et al., 2007). Although we have no additional evidence, it is plausible that DGKζ plays a role in the regulatory process for Mdm2 stability in the cytoplasm through as yet undetermined mechanisms.

Compelling evidence exists for an autoregulatory negative feedback loop to maintain p53 at low levels during normal homeostasis (Wu et al., 1993). Mdm2 is a major component consisting of an autoregulatory feedback system, in which p53 can control its own levels by inducing the expression of Mdm2 (Haupt et al., 1997; Kubbutat et al., 1997). In this regard, a recent report described that DGKζ is identified as a p53-inducible gene product in human cells overexpressing wild-type p53 (Vrba et al., 2008). Wild-type p53 is shown to induce transcription of DGKζ gene by binding to its promoter and by subsequent acetylation of promoter-associated histones H3 and H4, suggesting a genetic link between p53 and DGKζ. Therefore, results of this study support the idea that DGKζ cytoplasmic translocation can provide a novel negative feedback loop of p53 under stress conditions. Characteristically, DGKζ serves dual roles: Increased cytoplasmic pool of DGKζ functions as a scaffolding protein to facilitate p53 degradation, and attenuated nuclear DGKζ downregulates p53 transactivation activity, both of which repress p53-mediated cytotoxicity synergistically. Presumably, the p53-DGKζ system is an additional layer of the autoregulatory negative feedback loop, which is finely organized in case of pathological conditions.

Another interesting finding of this study is that p53 protein is strongly upregulated in the brain of DGKζ-deficient mice under normal conditions as well as after kainate-induced seizures. DGKζ-deficient mice develop normally with no obvious phenotype under physiological conditions, which contrasts sharply against Mdm2-deficient mice, which are embryonic lethal because of excessive apoptosis induced by increased levels of p53 protein and its activity (Jones et al., 1995; Montes de Oca Luna et al., 1995). Why are DGKζ-deficient mice viable and fertile with increased p53 levels in the brain even under normal conditions? One explanation is provided by our result (Fig. 8A): p53 transactivation activity is greatly attenuated in the absence of nuclear DGKζ, even though p53 protein is potently induced. Therefore, it is speculated that the p53 transcriptional activity is compromised in the absence of DGKζ despite increased p53 protein levels in the nucleus. Furthermore, nuclear-resident wild-type DGKζ and cytoplasmic DGKζ interact with p53, suggesting that DGKζ binds to p53 both in the nucleus and cytoplasm. In addition, DGKζ and p53 interact mutually in untreated cells and cells treated with DNA-damaging agent. Therefore, we propose the idea that DGKζ serves as a sentinel to control p53 both during normal homeostasis and in stress responses.

Some details of the mechanisms remain unaddressed: how is DGKζ involved in p53 transcriptional activity. Many factors are thought to contribute to p53-mediated transactivation, although a comprehensive understanding has not been reached because of its complexity and dynamic nature. Among various p53-associated proteins that participate in p53-mediated transcription, those that interact with the p53 DNA-binding domain are shown to exert selective influences on p53 target genes and outcomes (Vousden and Prives, 2009). Brn3A and Hzf selectively induce p53 activation of genes encoding cell cycle regulators such as p21 to facilitate cell cycle arrest, although apoptosis stimulating proteins for p53 (ASPPs) selectively activates the expression of apoptotic regulators such as PUMA, Bax and Noxa to promote cell death. These specificities are achieved through post-translational modifications that are dynamically deposited on p53 in a context-specific manner (Kruse and Gu, 2009). In this regard, we found that DGKζ also interacts predominantly with the DNA-binding region of p53 and that they interact mutually under normal conditions. Results obtained from reporter assay and immunoblot analysis (Fig. 8A) suggest that DGKζ knockdown or gene ablation attenuates p53 transcriptional activity on both pro-arrest target p21 and pro-apoptotic target Bax, but not selectively. Based on these findings, it is speculated that DGKζ participates in the basic machinery of p53 transcription, the nature of which remains to be characterized.

It should also be mentioned that DGKζ knockdown is shown to attenuate pro-apoptotic p53 target Bax levels (Fig. 8A) and simultaneously to enhance PARP-cleavage (Fig. 3D). These findings seem contradictory to each other. In this respect, there are several cell death pathways, although the detailed mechanism of mammalian apoptosis has not been elucidated. Of these, mitochondrial apoptotic pathway is known to be regulated by the balance between pro-apoptotic Bax and Bak and anti-apoptotic Bcl-2 (Martin, 2010). On the other hand, PARP-cleavage seems to be the final common pathway, which reflects more widespread proteolysis that is critical biochemical event early during the process of cell death (Kaufmann et al., 1993). Therefore, we need to further investigate the mechanism that leads to cell death under conditions where DGKζ is translocated to the cytoplasm and p53 is downregulated.

The results obtained in this study are summarized in Fig. 8B. Taken previous findings together, the picture that emerges is as follows: under normal conditions, DGKζ negatively regulates cell cycle progression in the nucleus in a kinase-dependent manner. Once translocated to the cytoplasm under stress conditions, this enzyme acts as a scaffolding protein to promote p53 degradation via the Mdm2-dependent ubiquitin–proteasome pathway, leading to attenuation of p53-mediated cytotoxicity. Absence of DGKζ from the nucleus suppresses p53 transactivation activity, although the prolonged absence leaves its original role in the nucleus unattended, which can render the neurons susceptible to the initiation of aberrant cell cycle re-entry under stress conditions. This reminds us of the fact that cells have a host of protective stress responses, most of which switch into execution mode during prolonged activation (Herrup and Yang, 2007). These responses are a double-edged sword that could be either protective or detrimental, depending on the cell type, characteristics of the stress applied, and its severity and duration. This might be the case with DGKζ cytoplasmic translocation in post-mitotic neurons. Additionally, there exist the other DGK isozymes that reside in the cytoplasm of neurons. Of these, DGKβ is reported to localize at the postsynaptic region of spines and regulate spine remodeling through actin filament assembly (Hozumi et al., 2008; Hozumi and Goto, 2012; Hozumi et al., 2009). Since short anoxic–hypoglycemic episodes, through NMDA receptor activation and calcium influx, are shown to result in a profound structural remodeling of synaptic networks, through growth, formation and elimination of spines and synapses (Jourdain et al., 2002), there may be functional cross-talk between cytoplasmic DGKζ and DGKβ in the synaptic remodeling under stress conditions.

Finally, an important question is raised as to whether DGKζ cytoplasmic translocation has a strong impact on proliferating cells. As shown in our data obtained for the HeLa cell model of DOX-induced DNA damage, cytoplasmic DGKζ can provide refractoriness to chemotherapy in cancer cells. Previous reports have described that, in addition to its role as a transcription factor, p53 elicits a transcription-independent pro-apoptotic response in the cytoplasm by localizing to the mitochondria and by activating a direct mitochondrial death program (Green and Kroemer, 2009; Marchenko et al., 2007). In this respect, our data show that cytoplasmic DGKζ anchors p53 to the cytoplasm, facilitates its degradation, and exerts an anti-apoptotic effect against DNA-damaging agents, which might contribute to the pathogenesis of malignancy in cancer cells. Further clinical studies must be conducted to ascertain whether cytoplasmic localization of DGKζ is an adverse prognostic factor.

Reagents

Cell culture reagents were obtained from Wako Pure Chemical Industries. MG132 and leptomycin B (LMB) were purchased from Sigma. Doxorubicin and kainate were from Wako.

Cell lines and cell culture

HeLa cervix carcinoma cells (RIKEN BRC), HEK293 embryonic kidney cells (RIKEN BRC), p53-deficient Saos2 osteosarcoma cells (ATCC) and U2OS osteosarcoma cells (ATCC) expressing wild-type p53 were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 U/ml). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Animals

Male C57BL/6W mice (6 weeks old, weighing 20–25 g) were purchased from Japan SLC Inc. All procedures were conducted in accordance with Yamagata University Guide for the Care and Use of Laboratory Animals and the permission of the Animals Subject Committee. Generation of DGKζ-deficient mice was as described previously (Regier et al., 2005; Zhong et al., 2003). Mice were treated with an intra-peritoneal injection of 25 mg/kg kainate and observed for seizure activity (Racine, 1972). Control mice were injected with saline alone. Mice were sacrificed for immunoblotting 24 h after kainate treatment.

Plasmids, RNAi and transfection

FLAG-tagged or GFP-tagged DGKζ, DGKζΔNLS lacking a NLS (Δ250–264), DGKζΔC, DGKζΔC1/3 and DGKζ1-280 were described as previously (Evangelisti et al., 2010; Goto and Kondo, 1996; Hozumi et al., 2003; Okada et al., 2011). Kinase dead mutant of DGKζ (DGKζ-KD) (Evangelisti et al., 2007), GFP-NAP1L1 and GFP-NAP1L4 (Okada et al., 2011) and GFP-DGKι (Ito et al., 2004) were constructed as described. ‘Kinase-dead’ of the kinase-dead mutant DGKζ-KD was demonstrated in our previous study (Evangelisti et al., 2007). DGKζΔNLS-KD was constructed by deleting a NLS from DGKζ-KD. Full-length p53 cDNA and its truncated forms, including N-terminus (p53-N, a.a. 1–101), DNA-binding domain (p53-DB, a.a. 102–292) and C-terminus (p53-C, a.a. 293–393), were amplified by PCR and subcloned into pEGFP-C3 (Clontech). All constructs were fully verified by DNA sequencing. The Plasmids for pp53-Luc and pAP1-Luc were purchased from Clontech (Pathway Profiling System, Clontech), and pRL-null were from TOYO INK. All constructs for transfection were purified with EndoFree Plasmid Maxi Kit (Qiagen). Double-stranded silencing RNAs directed against human DGKζ (siDGKζ-1, target sequence 5′-CTGGAGCGAGTCAGCGACATA-3′; siDGKζ-2, target sequence 5′-TCGCGTCAGCATGCACGACTA-3′) and human Mdm2 (target sequence 5′-AACCTGAAATTTATTCACATA-3′) and control siRNA (All stars negative control) were purchased from Qiagen. Human p53 siRNA (sip53, target sequence 5′-GCTTCGAGATGTTCCGAGAGCTGAA-3′) was from Invitrogen. Cells were transfected with various constructs or siRNA using LipofectAMINE2000 reagent (Invitrogen) according to the manufacturer's instructions.

Immunoblotting and antibodies

Cells treated with or without doxorubicin were lysed in lysis buffer consisting 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1% Triton X-100, and protease inhibitors cocktail (Sigma). Protein concentration was determined using BCA Protein Assay Reagent (Piece, Rockford, IL) according to the instruction manual. Equal amounts of protein lysate were applied in SDS-PAGE and electrophoretically transferred on PVDF membrane (Millipore). After blocking with 5% skim milk in PBS-T, the membrane were immunoblotted with primary antibodies against p53 (1∶1000; clone DO-1; Santa Cruz), p53 (1∶500; Ab-4; NeoMarkers), Mdm2 (1∶1000; clone Ab-1; Calbiochem), E2F1 (1∶1000; clones KH20 and KH95; Millipore), FLAG-M2 (1 µg/ml; Sigma), GFP (1∶5000; Clontech), NAP1L1 and NAP1L4 (Okada et al., 2011), Bax (1∶1000; sc-526; Santa Cruz), p21 (1∶1000; OP64; Calbiochem), cleaved PARP (1∶1000; #5625; Cell Signaling), ubiquitin (1∶1000; U5379; Sigma), β-actin (1∶5000; clone AC-15; Sigma), DGKζ [0.1 µg/ml, rabbit (Hozumi et al., 2003), guinea pig (Okada et al., 2011)]. Immunoreactive complexes were visualized using the chemiluminescent ECL+Plus western blotting detection system (GE Healthcare). Band intensities were quantified by densitometry using ImageJ (National Institutes of Health) (Nakano et al., 2012).

Immunoprecipitation

Cell lysates were lysed in the lysis buffer described above. After centrifugation for 10 min at 16,000 g at 4°C, the supernatants were transferred into a new tube and mixed with protein-G–Sepharose beads (GE Healthcare) for 2 h at 4°C to eliminate non-specific binding. Preclear supernatants were mixed with 4 µg anti-GFP or anti-FLAG antibody for 12 h at 4°C and incubated with protein-G–Sepharose beads for 5 h. After washing with lysis buffer four times, the immunocomplexes were boiled for 10 min in SDS sample buffer (New England Biolabs). The samples were applied to an SDS-PAGE gel and subjected to immunoblot analysis as above.

Immunofluorescence microscopy

Cells were grown on a coverglass. After washing with PBS, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton/PBS. Cells were incubated with 10% normal goat serum for blocking and incubated with anti-FLAG or anti-p53 antibody overnight at room temperature (RT) in a moist chamber. After washing with PBS three times, cells were incubated with anti-mouse IgG–Alexa-Fluor-596 (red) or anti-mouse IgG–Alexa-Fluor-674 (blue) for 1 h. Fluorescence images were taken under a Zeiss Axioplan 2 microscope equipped with a confocal laser-scanning unit (Carl Zeiss LSM510Meta).

Cell viability

Cell viability was assessed by Crystal Ciolet assay and WST-1 assay. For Crystal Violet assay, HeLa cells were grown in a 6-well plate. After 24 h transfection, cells were incubated in the presence or absence of doxorubicin (DOX, 0.2 µg/ml) for 24 h. After washing with ice-cold PBS twice, cells were fixed with ice-cold methanol for 10 min and incubated with 0.5% Crystal Violet solution for 10 min at RT. The plate was carefully rinsed in distilled water and dried at RT. For WST-1 assay, HeLa cells were seeded in 96-well plates at optimal density of 1×103 cells per well and transfected for 24 h. After incubation in the presence or absence of DOX as noted above, the cell proliferation reagent WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) was added to each well, as specified by the supplier (Roche Applied Science). After 4 h incubation, absorbance (A) was measured on a microplate reader (SUNRISE REMOTE, Wako) at 450 nm. Wells without cells containing complete medium and WST-1 reagent acted as blank. Viability index was calculated according to the formula: (ADOX+/ADOX−)×100.

Reporter assay

HeLa cells were co-transfected with either control siRNA (siCont) or siDGKζ along with p53 reporter pp53-Luc or pAP1-Luc and pRL (Renilla luciferase)-null plasmid (internal control) using LipofectAMINE2000. After 48 h transfection, cells were treated with DOX (0.2 µg/ml) for 12 h or left untreated. Cell lysates were collected and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (TOYO B-Net). All luciferase values were normalized to the Renilla luciferase values (internal control) and expressed as fold induction. Luminescence was measured using a luminescencer-Octa (Atto).

Statistical analysis

Data were expressed as means ± s.d. or SEM from three or more independent experiments. Statistical analysis was performed by Student's t-test at a significance level of P<0.01 (*) or n.s. (not statistically significant).

We thank Dr Kazuhiko Igarashi (Tohoku University) for advice on luciferase assays and Drs Yoshihiko Araki and Mitsuaki Yanagida (Juntendo University) for discussions.

Author contributions

T.T. and K.G. conceived and designed experiments and wrote the manuscript. T.T. conducted most of the experiments. M.O. and K.T. generated plasmid constructs. Y. Hozumi generated antibodies. M.K.T. prepared DGKzeta knockout mice. C.K., Y. Hamamoto, A.M.M., M.K.T., and M.I. contributed to the experimental design and scientific advice.

Funding

This work was supported by Grants-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan [grant numbers 20390050 and 24390044 to K.G.].

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