Background: VRK1 phosphorylates mitotic histone H3 at Thr-3 and Ser-10, but its negative regulator was not elucidated during interphase. Results: The macrodomain of macroH2A1 interacts with VRK1, and this suppresses enzymatic activity of VRK1 during interphase. Conclusion: Specific binding between VRK1 and macroH2A1 is required to regulate the cell cycle-dependent histone H3 phosphorylation. Significance: Understanding epigenetic regulation of histone H3 during the cell cycle is important in cancer development.

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.

Vaccinia-related kinase 1 (VRK1) is a crucial protein kinase for mitotic regulation. VRK1 is known to play a role in germ cell development, and its deficiency results in sterility. Here we describe that VRK1 is essential for the maintenance of spermatogonial stem cells. To determine whether VRK1 plays a role in these cells, we assessed the population size of undifferentiated spermatogonia. Flow cytometry analyses showed that the number of undifferentiated spermatogonia was markedly reduced in VRK1-deficient testes. VRK1 was highly expressed in spermatogonial populations, and approximately 66% of undifferentiated spermatogonia that were sorted as an Ep-CAM+/c-kit−/alpha-6-integrin+ population showed a positive signal for VRK1. Undifferentiated stem cells expressing Plzf and Oct4 but not c-kit also expressed VRK1, suggesting that VRK1 is an intrinsic factor for the maintenance of spermatogonial stem cells. Microarray analyses of the global testicular transcriptome and quantitative RT-PCR of VRK1-deficient testes revealed significantly reduced expression levels of undifferentiated spermatogonial marker genes in early postnatal mice. Together, these results suggest that VRK1 is required for the proliferation and differentiation of undifferentiated spermatogonia, which are essential for spermatogenic cell maintenance.

Vaccinia-related kinase 3 (VRK3) is known as a pseudokinase that is catalytically inactive due to changes in motifs that are essential for kinase activity. Although VRK3 has been regarded as a genuine pseudokinase from structural and biochemical studies, recent reports suggest that VRK3 acts as an active kinase as well as a signaling scaffold in cells. Here, we demonstrate that VRK3 phosphorylates the nuclear envelope protein barrier-to-autointegration factor (BAF) on Ser4. Interestingly, VRK3 kinase activity is dependent upon its N-terminal regulatory region, which is excluded from the determination of its crystal structure. Furthermore, the kinase activity of VRK3 is involved in the regulation of the cell cycle. VRK3 expression levels increase during interphase, whereas VRK1 is enriched in late G2 and early M phase. Ectopic expression of VRK3 induces the translocation of BAF from the nucleus to the cytoplasm. In addition, depletion of VRK3 decreases the population of proliferating cells. These data suggest that VRK3-mediated phosphorylation of BAF may facilitate DNA replication or gene expression by facilitating the dissociation of nuclear envelope proteins and chromatin during interphase.

Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases.

A pro-apoptotic function of activated PKCδ may be mediated by several downstream nuclear regulators involved in apoptotic cell death. Vaccinia-related kinase 1 (VRK1) is a new nuclear target of PKCδ in the regulation of apoptotic cell death induced by DNA damage.

Betulinic acid (BA) is a naturally arising pentacyclic triterpenoid that has anti-malarial, anti-retroviral, anti-inflammatory, and anti-cancer biological effects. More recently, it has been reported to possess anti-obesity activity mediated by the activation of AMP-activated protein kinase (AMPK). We further investigated antidiabetic activity of BA in mouse tissues at the cellular and systemic levels. We found that BA stimulated AMPK in a similar fashion to the known AMPK activators, such as 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside and metformin. Notably, the level of glucose uptake by BA was not altered by wortmannin, suggesting that this activation did not depend on phosphoinositide 3-kinase. Furthermore, BA diminished blood glucose levels in alloxane-treated ICR mice and in untreated mice during the glucose tolerance test. BA also stimulated mRNA expression of glucose transporter 4, which could partly explain increased glucose uptake. BA also increased AS160 phosphorylation by insulin-independent mechanisms in the extensor digitorum longus muscle. These results indicate that BA may serve as a promising therapeutic agent for diabetes by activating AMPK, like metformin. Notably, BA also enhanced mouse endurance capacity, indicating that it also affects metabolic regulation in addition to its antidiabetic activity.

Adrenal medulla chromaffin cells are neuroendocrine and modified sympathetic ganglion cells. Catecholamines released from chromaffin cells mediate the fight-or-flight response or alert reaction against dangerous conditions. Here we report that short-term treatment with glucocorticoids, released from adrenal cortex cells in response to chronic stress, inhibits activity-dependent potentiation (ADP) of catecholamine release. First, short-term treatment with dexamethasone (DEX), a synthetic glucocorticoid, reduces ADP in a concentration-dependent manner (IC50 324.2 ± 54.5 nm). The inhibitory effect of DEX is not reversed by RU-486 treatment, suggesting that the rapid inhibitory effect of DEX on ADP of catecholamine release is independent of glucocorticoid receptors. Second, DEX treatment reduces the frequency of fusion between vesicles and plasma membrane without affecting calcium influx. DEX disrupts activity-induced vesicle translocation and F-actin disassembly, thereby leading to inhibition of the vesicle fusion frequency. Third, we provide evidence that DEX reduces F-actin disassembly via inhibiting phosphorylation and translocation of myristoylated alanine-rich C kinase substrate and its upstream kinase protein kinase Cε. Altogether, we suggest that glucocorticoids inhibit ADP of catecholamine release by decreasing myristoylated alanine-rich C kinase substrate phosphorylation, which inhibits F-actin disassembly and vesicle translocation.