PKCδ regulates Mdm2 independently of p53 in the apoptotic response to DNA damage.

Apoptosis is the key process in which cells with defective genome can be eliminated. Dys-regulation of apoptosis causes accumulation of irreparable mutation arisen from DNA damage and is the underlying cause of carcinogenesis. PKCδ is a multifunctional kinase involved in signal transduction of genotoxic-induced apoptosis. Previous studies have demonstrated that PKCδ transactivates p53 in response to DNA damage. These findings led us to determine if Mdm2, a nuclear phospho-protein and negative regulator of p53, could also be a PKCδ-modulated substrate. We discovered that inhibition of PKCδ down-regulates Mdm2 protein expression regardless of p53 status. Given that Mdm2 mRNA change was detected in p53-proficient, but not deficient cells, PKCδ affected Mdm2 on the post-translational level. Interestingly, treatment of MG132 restored Mdm2 expression to the steady-state level. Further investigation showed that PKCδ inhibited Mdm2 ubiquitination in p53-deficient cells and loss of PKCδ resulted in an increase in Mdm2 proteosomal degradation. Moreover, P300/CBP-associated factor (PCAF), an ubiquitin ligase 3 for Mdm2, was observed to participate in Mdm2 ubiquitination by PKCδ inhibition and knock-down of PCAF rescued Mdm2 diminution. Finally, as shown for PKCδ, Mdm2 was also required to exert pro-apoptotic response caused by genotoxic agents in p53-null cells. In addition, overexpression of Mdm2 restored inhibitory effect of apoptosis in cells silenced for PKCδ. Taken together, we 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.

Identification of Evi-1 as a novel effector of PKCδ in the apoptotic response to DNA damage.

Protein kinase C delta (PKCδ), a PKC family isoform, regulates diverse signal transduction pathways during DNA damage to induce apoptosis. To explore the apoptosis mechanism that PKCδ modulates, we sought to uncover transcription factor targets of PKCδ by devising a screening strategy that utilizes ChIP-cloning and microarray analysis. Transcription factor candidates were generated with the application of public access data-mining tools and this resulted in the identification of Evi-1 as a novel PKCδ-mediated DNA damage responsive molecule. The results demonstrated that PKCδ is constitutively associated with Evi-1. PKCδ regulated Evi-1 to activate PLZF transcription upon genotoxic stress. Furthermore, both Evi-1 and PLZF were associated with DNA damage-stimulated apoptosis. Taken together, we have discovered a novel regulation of Evi-1, which transactivates PLZF, by PKCδ to induce cell death in response to genotoxic stress.

DNA damage signalling recruits RREB-1 to the p53 tumour suppressor promoter.

Transcriptional regulation of the p53 tumour suppressor gene plays an important role in the control of the expression of various target genes involved in the DNA damage response. However, the molecular basis of this regulation remains obscure. In the present study we demonstrate that RREB-1 (Ras-responsive-element-binding protein-1) efficiently binds to the p53 promoter via the p53 core promoter element and transactivates p53 expression. Silencing of RREB-1 significantly reduces p53 expression at both the mRNA and the protein levels. Notably, disruption of RREB-1-mediated p53 transcription suppresses the expression of the p53 target genes. We also show that, upon exposure to genotoxic stress, RREB-1 controls apoptosis in a p53-dependent manner. These findings provide evidence that RREB-1 participates in modulating p53 transcription in response to DNA damage.