Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21 WAF1 , MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21 WAF1 . In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.

NFBD1/MDC1, which belongs to the BRCT superfamily, has an anti-apoptotic activity and contributes to the early cellular responses to DNA damage. Here we found that NFBD1 protects cells from apoptotic cell death by inhibiting phosphorylation of p53 at Ser-15 under steady state as well as early phase of DNA damage, thereby blocking its transcriptional and pro-apoptotic activities. During late phase of DNA damage, a remarkable reduction of NFBD1 was observed in dying but not in surviving A549 cells bearing wild-type p53. Small interference RNA-mediated knockdown of the endogenous NFBD1 resulted in an increase in sensitivity to adriamycin in A549 cells but not in p53-deficient H1299 cells. Immunoprecipitation and luciferase reporter analyses demonstrated that NFBD1 binds to the NH 2 -terminal region of p53 and strongly inhibits its transcriptional activity. Additionally, BRCT domains, which can interact with p53, reduced the adriamycin-induced phosphorylation levels of p53 at Ser-15 and also suppressed the transcriptional activity of p53. Thus, our present findings strongly suggest that NFBD1 plays an important role in the decision of cell survival and death after DNA damage through the regulation of p53. The BRCA1 carboxyl terminus (BRCT)3 domain is defined by distinct hydrophobic clusters of amino acids and is often found in a variety of cellular proteins such as BRCA1, 53BP1, and RAD9, which are involved in DNA repair and/or DNA damage signaling pathways (1-3). Although the functional role of the BRCT domain is currently unclear, the early works indicated that the BRCT domains of BRCA1 act as a transactivator (4, 5). Consistent with this notion, the point mutations detected within the BRCT domains of BRCA1 markedly inhibited its transcriptional activity (4, 5), and BRCA1 was a component of RNA polymerase II holoenzyme (6). Alternatively, the BRCT domains function as protein-protein interaction modules (7). For example, the BRCT domains of BRCA1 as well as 53BP1 were required for the interaction with p53 (8, 9). NFBD1/MDC1 (nuclear factor with BRCT domain 1/mediator of DNA damage checkpoint protein 1) is a large nuclear protein bearing three characteristic structural domains, including an NH 2 -terminal forkhead-associated (FHA) domain, an internal PST (proline/serine/threonine-rich) repeat domain, and tandem repeat of COOH-terminal BRCT domains (10 -14). We have initially reported that NFBD1 acts as a nuclear transcriptional factor with an anti-apoptotic function (11). In accordance with our results, siRNA-mediated knockdown of NFBD1 led to a significant increase in the number of apoptotic cells (15). In addition, the elimination of NFBD1 expression increased the sensitivity to irradiation (16). These observations indicate that NFBD1 has an anti-apoptotic function; however, the precise molecular mechanisms behind the anti-apoptotic effect of NFBD1 remain to be explored.Previous studies strongly suggest that NFBD1 is closely involved in early cellular responses to genotoxic stress. Upon DNA damage, NFBD1 was phosp...

p73 protein level is kept extremely low in mammalian cultured cells and its stability may be regulated by not only the ubiquitin/proteasome-dependent proteolysis but also through other unidentified mechanisms. Here, we found for the first time that p73 is physically as well as functionally associated with the U-box-type E3/E4 ubiquitin ligase UFD2a. The immunoprecipitation experiments demonstrated that this interaction is mediated by the COOH-terminal region of p73alpha containing SAM domain. During the cisplatin-induced apoptosis in SH-SY5Y neuroblastoma cells, p73alpha accumulated at a protein level, whereas the endogenous UFD2a was significantly reduced in response to cisplatin. Ectopic expression of UFD2a decreased the half-life of p73alpha in association with a significant inhibition of the p73alpha-mediated transactivation as well as proapoptotic activity. Downregulation of endogenous UFD2a by antisense strategy resulted in a remarkable accumulation of p73alpha. Unexpectedly, UFD2a-mediated degradation of p73alpha was sensitive to the proteasomal inhibitor, however, UFD2a did not affect the ubiquitination levels of p73alpha. Taken together, our present findings imply that UFD2a might promote the proteasomal turnover of p73 in a ubiquitination-independent manner, and also suggest that UFD2a might play an important role in the regulation of cisplatin-induced apoptosis mediated by p73.

The extremely high rate (68.8%) of peritoneal metastases was observed in long-term follow-up for the metastatic breast cancer patients with ILC. We need to reveal the definitive feature of ILC and develop new therapeutic strategies to prevent the dissemination of ILCs.

I kappa B kinase (IKK) complex plays an important role in the regulation of signaling pathway that activates nuclear factor-kappa-B (NF-jB). Recently, we reported that cisplatin (CDDP) treatment causes a remarkable nuclear accumulation of IKK-a in association with stabilization and activation of p73. However, underlying mechanisms of CDDP-induced nuclear accumulation of IKK-a are elusive. Here, we found that ataxia-telangiectasia mutated (ATM) is one of upstream mediators of IKK-a during CDDP-induced apoptosis. In response to CDDP, ATM was phosphorylated at Ser-1981, which was accompanied with nuclear accumulation of IKK-a in HepG2 cells, whereas CDDP treatment had undetectable effects on IKK-a in ATM-deficient cells. Indirect immunofluorescence experiments demonstrated that phosphorylated form of ATM colocalizes with nuclear IKK-a in response to CDDP. In vitro kinase assay indicated that ATM phosphorylates IKK-a at Ser-473. Moreover, IKKa-deficient MEFs displayed CDDP-resistant phenotype as compared with wild-type MEFs. Taken together, our present results suggest that ATM-mediated phosphorylation of nuclear IKK-a, which stabilizes p73, is one of the main apoptotic pathways in response to CDDP.