Apoptotic Phosphorylation of Histone H3 on Ser-10 by Protein Kinase Cδ

Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC) δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new ‘apoptotic histone code’ mediated by PKCδ.


Introduction
Post-translational modifications of histone, including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, and adenosine diphosphate ribosylation, play important roles in the regulation of various cellular processes, such as gene expression, DNA replication and repair, chromatin condensation, chromosome segregation, and apoptotic cell death [1][2][3][4]. Histone tails are the primary sites of these various types of post-translational modifications and both determine the interaction between histones and other proteins and regulate chromatin structure [5]. The Nterminal tail of histone H3 is able to undergo several different types of epigenetic modification that influence cellular processes [6]. There are several well-characterized residues for phosphorylation within the N-terminal tail of histone H3: Thr-3, Ser-10, Thr-11, and Ser-28. Notably, the phosphorylation of histone H3 on Ser-10 in the N-terminal tail is essential for cell cycle progression and chromosome condensation during mitosis and meiosis [7]. A correlation between histone H3 Ser-10 phosphorylation and chromatin condensation during mitosis has been extensively studied [6,8]. However, phosphorylation of histone H3 on Ser-10 also occurs when cells are exposed to diverse death stimuli [9,10]. Although it has been reported that there is a correlation between histone H3 Ser-10 phosphorylation and chromosome condensation during apoptosis, the cellular effects of such phosphorylation remain elusive, and it has not been clearly elucidated whether this phosphorylation contributes to apoptotic cell death [11,12]. Additionally, there are no reports of specific protein kinases responsible for the phosphorylation of histone H3 on Ser-10 during apoptosis to date.
Protein kinase C (PKC), a family of serine/threonine protein kinases, is activated by various stimuli and involved in cellular processes, such as proliferation, survival, differentiation, and apoptosis [13,14]. The PKC family is comprised of at least 11 isozymes (a, bI, bII, c, d, e, f, g, h, i/l, and m) categorized according to activators, tissue distribution, and substrates. These PKC isozymes have been classified into three broad groups: i) the classical PKCs (cPKCs: PKCa, PKCbI, PKCbII, and PKCc), which are calcium-dependent and activated by diacylglycerol (DAG); ii) the novel PKCs (nPKCs: PKCd, PKCe, PKCg, and PKCh), which are calcium-independent and activated by DAG; and iii) the atypical PKCs (aPKCs: PKCf and PKCi), which are calcium-independent but not activated by DAG [15]. PKCd, a member of the nPKC family, is activated by diverse stimuli, including anti-cancer drugs, ionizing radiation, reactive oxygen species, growth factors, and cytokines [16]. In fact, many apoptotic stimuli can induce PKCd translocation to mitochondria, resulting in cytochrome C release and caspase-3 activation, as well as the generation of a constitutively active PKCd catalytic fragment [17]. Upon exposure of cells to DNA-damaging agents, PKCd undergoes caspase-3-mediated cleavage, resulting in the generation of catalytic fragment of PKCd. In most cells, catalytic fragment of PKCd is associated with apoptotic cell death. Ectopic expression of the catalytic fragment of PKCd is sufficient to induce apoptotic cell death [18,19]. In addition, PKCd-deficient cells are resistant to stresses induced by several stimuli, suggesting that PKCd is crucial for apoptotic cell death [20]. Therefore, PKCd is generally considered a growth inhibitory or pro-apoptotic kinase in mammalian cells.
Here, we show that phosphorylation of histone H3 on Ser-10 during apoptosis is mediated by the pro-apoptotic kinase PKCd. It is responsible for the phosphorylation of histone H3 on Ser-10 in vitro and in vivo. Ectopic expression of catalytically active PKCd robustly induced phosphorylation of histone H3 on Ser-10 and results in apoptotic cell death. We also demonstrate that activation of PKCd is required for histone H3 Ser-10 phosphorylation during apoptosis and thus contributes to chromatin condensation.

Results
PKCd Specifically Phosphorylates Histone H3 on Ser-10 in vitro In our attempt to find a novel histone kinase, we identified that PKCd robustly phosphorylates histone H3 in a protein kinase assay using radioactive adenosine triphosphate (data not shown). To elucidate whether PKCd is involved in the modification of the N-terminal tail of histone H3, we performed an in vitro kinase assay and analyzed specific phosphorylation by immunoblotting with several anti-phosphohistone H3-specific antibodies. The N-terminal tail of histone H3 is subject to diverse phosphorylation, including Thr-3 by haspin and VRK1, Seri-10 by Aurora B and VRK1, Thr-11 by Dlk/Zip kinase, and Ser-28 by Aurora B [21][22][23][24][25]. As shown in Figure 1A, PKCd specifically phosphorylated free core histones on Ser-10 of histone H3. As expected, the Ser-14 residue of histone H2B was also phosphorylated by PKCd [26]. Likewise, PKCd also phosphorylated Ser-10 when purified histone H3 was used as a substrate ( Figure 1B). Importantly, the ability of PKCd to phosphorylate histone H3 on Ser-10 is completely abolished when the residue is mutated to alanine ( Figure 1C).
In contrast, other known phosphorylation sites located in the Nterminal tail of histone H3, Thr-3 and Thr-11, were unaffected by PKCd when either core histone or free histone H3 were used as substrates ( Figure 1A and B). We also tested whether Ser-28 of histone H3 is also phosphorylated by PKCd, because both Ser-28 and Ser-10 are located immediately after the same amino acid motif-ARKS-in the tail of histone H3. As shown in Figure S1, PKCd phosphorylates Ser-28 of histone H3 when both core histone and free histone H3 were used as substrates. PKCd phosphorylates histone H3 at Ser-10 and Ser-28 in vitro simultaneously as does Aurora B.

Catalytically Active PKCd Phosphorylates Histone H3 on Ser-10 in Cells
To explore the physiological role of PKCd in mediating histone H3 Ser-10 phosphorylation, we transfected HEK293T cells with plasmids expressing either a constitutively active catalytic fragment (CF) or a kinase-dead dominant-negative (CF-DN) PKCd mutant ( Figure 2A). Ectopic expression of PKCd CF dramatically increased the level of phosphorylation on Ser-10 of histone H3, whereas there was no substantial change in cells expressing PKCd CF-DN ( Figure 2A). Catalytically active PKCd significantly enhances the level of phosphorylation on Ser-14 of histone H2B, a typical marker of chromatin condensation during apoptosis. Under the above conditions, apoptotic cell death signaling was evident from the increased amount of cleaved caspase-3 ( Figure 2A). In accordance with the in vitro kinase assay shown in Figure 1 A and B, there were no remarkable changes in phosphorylation on Thr-3 or Thr-11 of histone H3 when HEK293A cells were transfected with catalytically active PKCd ( Figure 2A). However, it is noteworthy that there were no significant increases in the levels of phosphorylation on Ser-28 of histone H3 in PKCd-overexpressing cells ( Figure 2A). These data indicate the possibility that histone H3 Ser-28 is not the main phosphorylation site by PKCd under physiological conditions.
To demonstrate whether PKCd triggers the phosphorylation of histone H3 on Ser-10 during apoptosis, we stained cells with phospho-H3 Ser-10-specific antibody. Cells transfected with PKCd CF induced remarkable histone H3 Ser-10 phosphorylation but not in PKCd CF-DN-or mock vector-transfected cells ( Figure 2B). Strikingly, catalytically active PKCd elicited histone H3 Ser-10 phosphorylation in greater than 80% of transfected cells ( Figure 2C). Collectively, these observations suggest that activation of PKCd is sufficient to promote the phosphorylation on Ser-10 of histone H3 during apoptotic cell death.

Phosphorylation of Histone H3 on Ser-10 by PKCd during Apoptotic Cell Death
To investigate the physiological relevance between histone H3 Ser-10 phosphorylation and apoptotic cell death, we tested whether the histone modification occurred in cells during DNA damage-induced apoptosis. To examine the role of histone H3 Ser10 phosphorylation in apoptotic cell death, it is necessary to exclude mitotic histone H3 Ser10 phosphorylation. To do this, we synchronized the cells to G1 to exclude the phosphorylation of H3 on Ser10 in mitosis. Jurkat cells ( Figure 3A) or HeLa cells ( Figure 3B) were pretreated with hydroxyurea for 24 hrs before the addition of cisplatin to elicit DNA damage-induced apoptosis. As expected, cells arrested in the G1 phase after treatment with hydroxyurea showed decreased levels of phosphorylation on Ser-10 of histone H3 ( Figure 3A and B). After cell cycle synchronization, the treatment of cells with cisplatin triggered the induction of major apoptotic markers, such as cleaved caspase-3 and PKCd CF. Additionally, the phosphorylation of histone H3 on Ser-10 was also increased, suggesting that PKCd is the kinase responsible for the phosphorylation of histone H3 on Ser-10 that occurs during programmed cell death ( Figure 3A and B). To confirm whether PKCd is responsible for histone H3 Ser-10 phosphorylation during DNA damage-induced apoptosis, we knocked down PKCd by transfection of Jurkat cells with PKCd siRNAs. Downregulation of PKCd abrogated histone H3 Ser-10 phosphorylation induced by cisplatin ( Figure 3C). Moreover, the induction of cleaved caspase-3 was diminished (Fig. 3C). On the basis of MTT assay, we also found that depletion of PKCd resulted in resistance to apoptotic cell death following cisplatin treatment as well as reduction of histone H3 Ser10 phosphorylation ( Fig 3D). These data suggest that PKCd-mediated histone H3 Ser10 phosphorylation is also crucial in apoptotic cell death. Besides, to investigate whether PKCd specifically phosphorylates histone H3 Ser10, we tested other PKC isoenzymes. When PKCa and PKCe were depleted, the phosphorylation of histone H3 on Ser10 were not affected by cisplatin treatment, suggesting that this phosporylation is solely induced by PKCd in DNA damage-induced apoptosis ( Figure S2).
To demonstrate the involvement of endogenous PKCd in the phosphorylation of histone H3 at Ser10 during apoptotic cell death, cisplatin-treated HeLa cells were examined by immunofluoresence microscopy with anti-PKCd and anti-phosphohistone H3 Ser10-specific antibody after cell cycle arrest induced by hydroxyurea. As shown in Figure 3E (upper panel), cytoplasmic localization of PKCd was prominent in most untreated control cells. In addition, PKCd antibody did not stain the mitotic cell chromosomes which was positive for the phospho-histone H3 Ser10 (see arrows), confirming that PKCd is not associated with mitotic chromosomal condensation. In contrast, when HeLa cells were treated with cisplatin after cell cycle synchronization ( Figure 3E, lower panel), activated PKCd translocated to the nucleus and co-localized with the phospho-histone H3 Ser10 positive cells(see arrows). This immunocytochemical analysis suggests that apoptotic phosphorylation of histone H3 on Ser10 is associated with the activation and translocation of PKCd in DNA damage-induced apoptosis. To investigate the nuclear translocation and colocalization of PKCd with phospho-histone H3 Ser 10 in DNA damage-induced apoptosis, we also performed co-immunoprecipitation assay before and after exposing the cells to cisplatin. We could detect endogenous interaction between activated PKCd and histone H3 only under DNA-damaging conditions, suggesting that they could associate with each other in process of apoptotic cell death ( Figure 3F).
Furthermore, cisplatin-treated HeLa cells were also analyzed by both terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytochemical analysis using anti-phosphohistone H3 Ser10-specific antibody. Surprisingly, we found that about 10% of TUNEL-positive cells were also positive for the phosphorylation of histone H3 on Ser10 ( Figure 4A and Figure S3). In addition, cleaved caspase 3positive cells were also labeled with anti-phosphohistone H3 Ser10 antibody ( Figure 4B). Hence, these data indicate that PKCd mediates the phosphorylation of histone H3 on Ser10 during apoptotic cell death.
To test whether activated PKCd is involved in chromosome condensation during apoptosis, HEK293A cells were transfected with enhanced green fluorescent protein (EGFP), EGFP-PKCd CF, and EGFP-PKCd CF-DN plasmids, respectively. The transfected cells were labeled with anti-phosphohistone H3 Ser-10 antibody, and the nuclei were stained with Hoechst 33342. As shown in Figure 2B, cells transfected with PKCd CF also stain positive for histone H3 Ser-10 phosphorylation. These cells also contain shrunk and dense nuclear bodies by Hoechst staining, compared to non-transfected cells ( Figure 5, middle panel, and white arrow). In contrast, cells transfected with mock vector or PKCd CF-DN did not show any remarkable changes in nuclear morphology ( Figure 5, upper and lower panels). These data suggest that PKCd phosphorylates histone H3 on Ser-10 as well as histone H2B on Ser-14, triggering the chromatin condensation required for apoptotic cell death.

Discussion
An increasing number of reports have found that posttranslational modifications of histones affect overall chromatin structure and result in the regulation of nuclear function, leading to the formation of the 'histone code' hypothesis [29,30]. According to this hypothesis, different combinations of histone modifications are connected to specific chromatin functions and nuclear processes during DNA repair, mitosis, meiosis, development, and apoptosis. Among the various histone modifications, specific phosphorylation sites within histone tails have been thought to affect chromatin structure and function during apoptotic cell death. It has been clearly demonstrated that phosphorylation of histones H2A, H2B, H3, and H4 is associated with nuclear events in cell death, providing cumulative evidence for an 'apoptotic histone code' or ''death code [12,31].'' In the present study, we report that PKCd is responsible for the phosphorylation of histone H3 on Ser-10 during apoptosis and contributes to the formation of chromatin condensation featured during programmed cell death. Therefore, this study suggests that histone H3 Ser-10 has a role in apoptosis as well as in mitosis. The pro-apoptotic role of catalytically activate PKCd is mediated by several downstream nuclear proteins associated with the induction of apoptosis. One of these proteins is the DNAdependent protein kinase, an enzyme essential for the repair of double-stranded DNA breaks, which is inhibited by PKCddependent phosphorylation [18]. Rad9, a key component of the genotoxin-activated checkpoint signaling complex, also binds to anti-apoptotic Bcl-2 family proteins and mediates responses to DNA damage when phosphorylated by PKCd during apoptosis [32]. With regard to histone modification during apoptosis, it has been reported that acinus-dependent PKCd activation increases histone H2B phosphorylation on Ser-14, which is closely associated with chromatin condensation during apoptosis [26,33]. In addition, PKCd also phosphorylates histone H3 on Thr-45 during apoptosis of HL60 and human neutrophil cells [34]. It has been proposed that the phosphorylation of histone H3 on Thr-45 by PKCd causes structural changes within the nucleosome to promote DNA fragmentation during late apoptosis [12]. These recent findings collectively support the notion that activated PKCd functions as the major histone kinase responsible for chromatin condensation and DNA fragmentation during programmed cell death. In this report, we demonstrate that PKCd actively phosphorylates histone H3 on Ser-10 both in vitro and in vivo (Figure 1 and 2). It is noteworthy that PKCd phosphorylates histone H3 on Ser-10 and histone H2B on Ser-14 at the same time. These results suggest that PKCd is the histone kinase responsible for histone H3 Ser-10 phosphoryation and thus contributes to chromatin condensation in combination with other 'death codes' during programmed cell death.
It is extensively known that mitotic chromatin condensation is closely related to histone H3 phosphorylation on Ser-10 [35]. It is also known that there is a correlation between histone H3 Ser-10 phosphorylation and chromatin condensation during apoptosis. However, this modification has not been clearly demonstrated during cell death to date. As the phosphorylation of Ser-10 of histone H3 accompanies chromatin condensation, a similar process might occur during apoptotic cell death. We also found that activation of PKCd was associated with the phosphorylation of histone H3 on Ser-10 during apoptosis and chromatin condensation in cells undergoing programmed cell death. Thus, it is probable that histone H3 Ser-10 phosphorylation mediates chromatin condensation during both apoptosis and mitosis. Therefore, this study provides the evidence of two distinctive functions mediated by the modification of histone H3 on Ser-10 that occurs during both apoptosis and mitosis.
In summary, we propose the following distinctive model of the phosphorylation of histone H3 on Ser-10 both during mitosis and apoptosis ( Figure 6). It is known that when cells are in a normal proliferative state, mitotic kinases such as Aurora B and VRK1 phosphorylate histone H3 on Ser-10 to promote chromatin condensation during mitosis [22,23]. In contrast, when cells are exposed to apoptotic stimuli, activated PKCd phosphorylates histone H3 on Ser-10 to facilitate chromatin condensation during apoptosis ( Figure 6). In conclusion, histone H3 Ser-10 phosphorylation can be designated as a new 'apoptotic histone code' mediated by PKCd.

DNA Constructs
PKCd cloned into pHACE vector was generously given by Dr. Y. S. Lee. [2] The constitutive active form of PKCd (amino acids 325-674, catalytic fragment, CF) was prepared by subcloning of the PCR-amplified fragment into the Xho I and Bam HI sites of pEGFP-C1 (Clontech). PKCd in vitro Kinase Assay Recombinant PKCd was purchased from Upstate Biotechnology. The kinase assay was carried out in a total volume of 30 ml of a kinase reaction buffer containing 50 mM ATP, and affinitypurified GST fusion proteins or 200 mg/ml bovine purified histone H3 (Roche) as substrate for 30 min incubation at 30uC. The phosphorylated proteins were resolved on 10% SDS-polyacrylamide gels followed by immunoblot analysis.

Cell Culture and Transfection
HEK293A or HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics in a humidified 5% CO 2 incubator at 37uC. Jurkat cells were grown in RPMI 1640 containing 10% fetal bovine serum, glutamine, HEPES, and antibiotics in a humidified 5% CO 2 incubator at 37uC. For the induction of apoptosis, cells were treated with 100 mM cisplatin (Sigma-Aldrich). For cell cycle synchronization, cells were treated with 1 mM hydroxyurea (Sigma-Aldrich) for 24 hours. HEK293A cells were transfected with the plasmid indicated in the specific experiments by using Lipofectamine 2000 (Invitrogen). After incubation for 24 h, the transfected cells were treated as indicated for analysis.

Immunocytochemical Analysis
Cells were grown on coated glass coverslips. For immunocytochemistry, cells were fixed with 4% PFA and then incubated with blocking solution (2.5% bovine serum albumin and 2.5% equine serum in PBS) for 1 h at room temperature. The samples were incubated overnight at 4uC with anti-phospho histone Ser 10 antibody (Abcam), followed by incubation with Alexa546-conjugated anti-rabbit IgG. Slides were mounted and visualized by fluorescence microscopy (Axioplan2, Zeiss; Oberkochen, Germany).

TUNEL Assay
For the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, HEK293A or HeLa cells were grown on glass chips coated with poly-D-lysine (Sigma). Cells were then fixed with 4% paraformaldehyde, the glass chips were removed, and the cells were permeabilized and stained with a DeadEnd fluorometric TUNEL system kit (Promega) according to the manufacturer's instructions.

Statistical Analysis
All quantitative data are presented as means6SD of a minimum of three experiments. Comparisons between two groups were analyzed via t test, and values of P,0.05 were considered to be significant.

Author Contributions
Conceived and designed the experiments: CHP. Performed the experiments: CHP. Analyzed the data: CHP KTK. Wrote the paper: CHP KTK.