**5. Role for p53 in response to DNA damage**

The tumor suppressor protein p53 plays a central role in mediating stress and DNA damageinduced cell cycle arrest and apoptosis (Vogelstein et al. 2000). The p53 protein controls normal responses to DNA damage and other forms of genotoxic stress and is an indispensable element in maintaining genomic stability (Vogelstein et al. 2000). In fact, *p53* is the most frequently mutated gene in human cancers (Nigro et al. 1989). The level of p53 protein is mostly undetectable in normal cells but rapidly increases in response to a variety of stress stimuli. The mechanism by which the p53 protein is stabilized is not completely understood, but post-translational modification plays a crucial role (Shieh et al. 1997). Mutations in the *p53* gene are frequently correlated with generation of human cancers; however, the p53 pathway can be also derailed by diverse oncogenic molecules (Oren et al. 2002). The *p53* gene knockedout mice develop tumors with an increased rate (Donehower et al. 1992). It is reasonable that many agents may inhibit the p53 pathway as part of the road toward tumor promotion. However, mechanisms for action of many chemical agents that promote tumor development have not been elucidated. With the central role of p53 in mind, agents that promote tumor formation might block the p53 pathway. Importantly, p53 is regulated primarily via posttranslational modifications, especially phosphorylation, and the accumulation of p53 is the first step following cellular stress (Oren 1999). The *mdm2* gene is a transcriptional target of p53, and once synthesized, the MDM2 protein can bind to p53 at its NH2 terminus leading to its rapid degradation through the ubiquitin proteasome-mediated pathway (Kubbutat and Vousden 1998, Oren 1999, Ryan et al. 2001). Upon DNA damage, p53 is phosphorylated at multiple sites at the NH2 terminus, thereby inhibiting MDM2 binding (Burns and El-Deiry 1999, Canman et al. 1998, Kubbutat and Vousden 1998, Oren 1999, Ryan et al. 2001, Siliciano et al. 1997). As a result, p53 degradation stops and p53 accumulates. p53 can also be phosphorylated at its COOH-terminal regulatory domain, which influences its DNA binding

Role for PKCδ on Apoptosis in the DNA Damage Response 297

Recent study also demonstrated that PKC induces the promoter activity of p53 via the p53 core promoter element (CPE-p53) and that such induction is enhanced after DNA damage. Upon genotoxic insults, PKC is activated and interacts with the death-promoting transcription factor Btf (Bcl-2-associated transcription factor) to co-occupy CPE-p53. Inhibition of PKC decreases the affinity of Btf to CPE-p53, thereby reducing *p53* expression. Concomitant with these results, abrogation of Btf-mediated *p53* transcription by RNA interference leads to repression of p53-mediated apoptosis in response to genotoxic stress. These findings demonstrate that activation of *p53* transcription by PKC induces p53-

Recent study demonstrated that both PKC and IKK, but not IKK, are targeted to the nucleus after oxidative stress (Yamaguchi et al. 2007a, Yamaguchi et al. 2007b). PKC interacts with and activates IKK. Significantly, upon exposure to oxidative stress, PKC mediated IKK activation does not contribute to NF-B activation; rather, nuclear IKK controls transcription activity of p53 by phosphorylation on Ser20. These findings indicate a novel mechanism in which the PKCIKK signaling pathway contributes to ROS-induced p53 activation. Recent studies have also demonstrated that phosphorylation of p53 at Ser46 induces p53AIP1 expression, resulting in the commitment to the apoptotic cell death (Matsuda et al. 2002, Oda et al. 2000, Taira et al. 2007, Yoshida 2008b). Furthermore, upon genotoxic stress, p53DINP1 is induced and then recruits a kinase(s) to p53, which specifically phosphorylate Ser46 (Okamura et al. 2001). We initially found that PKC is associated with Ser46 phosphorylation (Yoshida et al. 2006a). This phosphorylation was required for the interaction of PKC to p53. Importantly, p53DINP1 associated with PKC in response to anti-cancer agents. In concert with these findings, PKC potentiates p53 dependent apoptotic cell death by Ser46 phosphorylation. Taken together, PKC controls p53 to induce apoptosis in the cellular response to DNA damage (Yoshida et al. 2006a). Of note, our subsequent studies have demonstrated that another kinase DYRK2 plays a major and direct role on apoptosis induction by phosphorylating p53 at Ser46 in response to DNA damage (Taira et al. 2007, Taira et al. 2010). We also recently found that PKC regulates MDM2 expression independently of p53. Given that *Mdm2* mRNA change was detected in p53-proficient, but not deficient cells, PKCδ affected Mdm2 at the post-translational level. In this context, treatment of proteasome inhibitor MG132 restored Mdm2 expression to the steady-state level. Moreover, PKCδ inhibited Mdm2 ubiquitination in p53-deficient cells and loss of PKCδ resulted in an increase in Mdm2 proteasomal degradation. P300/CBPassociated factor (PCAF), an ubiquitin ligase 3 for Mdm2, was observed to participate in Mdm2 ubiquitination by PKCδ inhibition and PCAF silencing rescued Mdm2 diminution. We thus conclude that PKCδ regulates Mdm2 expression distinctively of p53 pathway by affecting Mdm2 ubiquitination and maintenance of Mdm2 expression by PKCδ is important to ensure normal genotoxic cell death response in human cancer cells (Hew et al. 2011).

PKC plays a pivotal role in the control of apoptotic cell death in response to a diverse array of stress stimuli. Thus PKC is a pro-apoptotic kinase activated by multiple mechanisms, including subcellular translocation and proteolysis. The proteolytic activation of PKC is also important not only in activating the downstream apoptotic cascade including p53, but

dependent apoptosis following DNA damage (Liu et al. 2007).

**6.2 Control at the post-translation** 

**7. Future perspective** 

(Meek 1998). In this context, constitutive phosphorylation of p53 by PKC at its COOH-terminal domain can lead to its degradation through ubiquitin proteasome-mediated pathway (Chernov et al. 2001). Moreover, treatment with PKC inhibitors, such as H7 or bisindolylmaleimide I, prohibited COOH-terminal phosphorylation of p53 and increased accumulation of p53 without any effect on the formation of the p53-MDM2 complex (Chernov et al. 2001). However, PKC inhibitors were incapable of p53 accumulation in human papilloma virus-positive HeLa cells (Chernov et al. 2001, Chernov et al. 1998).
