Expression of the TP53 tumor suppressor is tightly controlled for its ability to function as a critical regulator of cell growth, proliferation, and death in response to DNA damage. However, little is known about the mechanisms and contributions of the transcriptional regulation of TP53. Here we report that protein kinase C ␦ (PKC␦), a ubiquitously expressed member of the novel subfamily of PKC isoforms, transactivates TP53 expression at the transcriptional level. Reporter assays demonstrated that PKC␦ induces the promoter activity of TP53 through the TP53 core promoter element (CPE-TP53) and that such induction is enhanced in response to DNA damage. The results also demonstrate that, upon exposure to genotoxic stress, PKC␦ activates and interacts with the death-promoting transcription factor Btf to co-occupy CPE-TP53. Inhibition of PKC␦ activity decreases the affinity of Btf for CPE-TP53, thereby reducing TP53 expression at both the mRNA and the protein levels. In concert with these results, we show that disruption of Btf-mediated TP53 gene transcription by RNA interference leads to suppression of TP53-mediated apoptosis following genotoxic stress. These findings provide evidence that activation of TP53 gene transcription by PKC␦ triggers TP53-dependent apoptosis in response to DNA damage.Apoptosis is a genetically controlled process of cell suicide, essential for the elimination of damaged cells from multicellular organisms. Inappropriate activation or inhibition of apoptosis contributes to the pathogenesis of cancer and autoimmune diseases (7, 16). However, the signaling mechanisms responsible for the regulation of apoptosis remain largely unclear. Protein kinase C ␦ (PKC␦), a ubiquitously expressed member of the novel subfamily of PKC isoforms, has emerged as a common mediator of apoptosis in response to many stimuli (19, 37). Previous studies have shown that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate-or oxidative stress-induced mitochondrial translocation of PKC␦ is associated with the loss of mitochondrial transmembrane potential and the release of cytochrome c (17, 18). These findings suggest that PKC␦ is involved in a mitochondrion-dependent apoptotic pathway. Other studies have demonstrated that PKC␦ interacts with c-Abl tyrosine kinase upon exposure to genotoxic stress (50). c-Abl is a proapoptotic tyrosine kinase that targets to the nucleus following genotoxic stress and oxidative stress (42,46,49). Importantly, activated PKC␦, by c-Abl-mediated phosphorylation, increases c-Abl activity simultaneously through interaction with c-Abl (28). In addition, PKC␦ phosphorylates SHP-1 tyrosine phosphatase and decreases its activity (40). Given that c-Abl is negatively regulated by SHP-1 in response to DNA damage (14), PKC␦ regulates apoptotic signals of the key apoptotic tyrosine kinase c-Abl not only by its activation but also by inhibiting the activity of SHP-1 tyrosine phosphatase. Furthermore, recent studies have demonstrated that PKC␦ interacts with and phosphorylates Rad9, a key factor involv...
Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage. microarray | Drosha | miRNA processing
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.
Nuclear factor-KB (NF-KB) is tightly modulated by IKB kinases and IKBA in the cytoplasm. On stimulation, NF-KB translocates into the nucleus to initiate transcription; however, regulation of its transcriptional activity remains obscure. Here, we show that protein kinase C (PKC) D controls the main subunit of NF-KB, RelA/p65. On exposure to tumor necrosis factor-A (TNF-A), the expression of RelA/ p65 target genes such as IKBA, RelB, and p100/p52 is upregulated in a PKCD-dependent manner. The results also show that PKCD is targeted to the nucleus and forms a complex with RelA/p65 following TNF-A exposure. Importantly, kinase activity of PKCD is required for RelA/p65 transactivation. In concert with these results, PKCD activates RelA/p65 for its occupancy to target-gene promoters, including IKBA and p100/p52. Moreover, functional analyses show that inhibition of PKCD is associated with substantial attenuation of NF-KB activity in response to TNF-A. These findings provide evidence that PKCD orchestrates RelA/p65 transactivation, a requisite for NF-KB signaling pathway in the nucleus.
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