**6.1 Control at the transcription**

Recent reports document that PKC transactivates expression of *p53* at the transcriptional level (Abbas et al. 2004, Liu et al. 2007, Yoshida 2008a). The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) hinders DNA damage-induced up-regulation of p53 by down-regulating PKC. TPA initiates tumor formation in a variety of mice and tissue culture models, and this has been correlated with the down-regulation of PKC (Hansen et al. 1990). TPA initially induces and then diminishes the activity of the diacylglycerol-dependent PKC isoforms (Fournier and Murray 1987, Hansen et al. 1990). Previous studies showed that the tumor-promoting activities of TPA are mediated at least in part by down-regulating PKC (Lu et al. 1997). Moreover, transgenic mice over-expressing PKC were resistant to tumor promotion by TPA (Reddig et al. 1999). In this regard, previous studies implied that TPA can inhibit the DNA damage-mediated induction of p53 (Magnelli et al. 1995). Moreover, other studies with protein kinase inhibitors suggested that PKC regulates the p53 signalsome pathway (Ghosh et al. 1999). Regulation of p53 upon stress most commonly occurs by inhibiting ubiquitination and degradation of the p53 protein. In contrast, repression of p53 by inhibiting PKC is caused by the prevention of p53 synthesis, not augmented degradation of p53 protein. Inhibiting PKC blocks both basal transcription of the human *p53* gene and initiation of transcription from the human *p53* promoter. The DNA damage-elicited increase in *p53* accumulation is drastically inhibited by pre-treatment with TPA. In addition, the PKC inhibitor, rottlerin, is also able to block the DNA damage-mediated induction of *p53*. More importantly, pre-treatment of cells with TPA or treatment with rottlerin results in the inhibition of basal *p53* transcription. In this regard, accumulation of *p53* could not be achieved by any means, including proteasome inhibition, after TPA or rottlerin treatment, since *p53* transcription is hindered. Thus, the tumorsuppressing effects for PKC are mediated at least in part through activating *p53* transcription. Suppression of the *p53* promoter has been implied as a mechanism for tumor promotion (Raman et al. 2000, Stuart et al. 1995). Damaged genes in tumor cells are generally the mechanistic drivers toward oncogenesis. However, abrogation of endogenous genes, specifically tumor suppressors, may be also a crucial regulatory mechanism for tumor promotion. In this context, agents that interfere with the activity of PKC may inhibit p53 responses.

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

#### **6.2 Control at the post-translation**

296 Selected Topics in DNA Repair

(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

The p53 tumor suppressor is activated following genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or apoptosis. Accumulating studies have demonstrated that PKC regulates p53 expression at the transcriptional and post-

Recent reports document that PKC transactivates expression of *p53* at the transcriptional level (Abbas et al. 2004, Liu et al. 2007, Yoshida 2008a). The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) hinders DNA damage-induced up-regulation of p53 by down-regulating PKC. TPA initiates tumor formation in a variety of mice and tissue culture models, and this has been correlated with the down-regulation of PKC (Hansen et al. 1990). TPA initially induces and then diminishes the activity of the diacylglycerol-dependent PKC isoforms (Fournier and Murray 1987, Hansen et al. 1990). Previous studies showed that the tumor-promoting activities of TPA are mediated at least in part by down-regulating PKC (Lu et al. 1997). Moreover, transgenic mice over-expressing PKC were resistant to tumor promotion by TPA (Reddig et al. 1999). In this regard, previous studies implied that TPA can inhibit the DNA damage-mediated induction of p53 (Magnelli et al. 1995). Moreover, other studies with protein kinase inhibitors suggested that PKC regulates the p53 signalsome pathway (Ghosh et al. 1999). Regulation of p53 upon stress most commonly occurs by inhibiting ubiquitination and degradation of the p53 protein. In contrast, repression of p53 by inhibiting PKC is caused by the prevention of p53 synthesis, not augmented degradation of p53 protein. Inhibiting PKC blocks both basal transcription of the human *p53* gene and initiation of transcription from the human *p53* promoter. The DNA damage-elicited increase in *p53* accumulation is drastically inhibited by pre-treatment with TPA. In addition, the PKC inhibitor, rottlerin, is also able to block the DNA damage-mediated induction of *p53*. More importantly, pre-treatment of cells with TPA or treatment with rottlerin results in the inhibition of basal *p53* transcription. In this regard, accumulation of *p53* could not be achieved by any means, including proteasome inhibition, after TPA or rottlerin treatment, since *p53* transcription is hindered. Thus, the tumorsuppressing effects for PKC are mediated at least in part through activating *p53* transcription. Suppression of the *p53* promoter has been implied as a mechanism for tumor promotion (Raman et al. 2000, Stuart et al. 1995). Damaged genes in tumor cells are generally the mechanistic drivers toward oncogenesis. However, abrogation of endogenous genes, specifically tumor suppressors, may be also a crucial regulatory mechanism for tumor promotion. In this context, agents that interfere with the activity of PKC may inhibit

virus-positive HeLa cells (Chernov et al. 2001, Chernov et al. 1998).

**6. PKC regulation of p53** 

**6.1 Control at the transcription** 

translational levels.

p53 responses.

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).
