Protein Kinase Cδ-mediated Phosphorylation of Vaccinia-related kinase 1 and Histone H3 in DNA Damage-induced Apoptosis

Abstract

Protein kinase Cδ (PKCδ) is a serine/threonine kinase that plays a crucial role in the regulation of diverse cellular processes, including cell proliferation, differentiation and apoptosis. Since the discovery that PKCδ is a substrate for caspase-3, there has been numerous reports that connected PKCδ with pro-apoptotic signaling. However, the nuclear targets pro-apoptotic PKCδ and its signaling mechanisms that promote apoptotic cell death are largely unknown. Here we report that pro-apoptotic PKCδ phsophorylates vaccinia-related kinase 1 (VRK1) and histone H3 on Ser-10 in DNA damage induced apoptosis. VRK1 is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase C (PKC) in the regulation of VRK1. We show that PKC interacts with VRK1, phosphorylates the Ser355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to DNA damaging agent. In addition, p53 played a critical role in the regulation of DNA damage-induced cell death accompanied by PKCd-mediated modulation of VRK1. In p53-deficient cells, PKCd-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCd. Taken together, these results indicate that PKC regulates phosphorylation and downregulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. 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.