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.

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.

p73 shares high sequence homology with the tumor suppressor p53. Like p53, ectopic overexpression of p73 induces cell cycle arrest and/or apoptosis, and these biological activities are linked to its sequence-specific transactivation function. The COOH-terminal region of p73 is unique and has a function to modulate DNA-binding ability and transactivation activity. To identify and characterize cellular proteins that interact with the COOH-terminal region of p73␣ and regulate its activity, we employed a yeast-based two-hybrid screen with a human fetal brain cDNA library. We found MM1, a nuclear c-Myc-binding protein, was associated with p73␣ in both yeast two-hybrid and in vitro pull-down assays. In mammalian cells, MM1 co-immunoprecipitated with p73␣, whereas p73␤ and tumor suppressor p53 did not interact with MM1. Overexpression of MM1 in p53-deficient osteosarcoma SAOS-2 cells enhanced the p73␣-dependent transcription from the p53/p73-responsive Bax and PG13 promoters, whereas p73␤-and p53-mediated transcriptional activation was unaffected in the presence of MM1. MM1 also stimulated the p73␣-mediated growth suppression in SAOS-2 cells. More importantly, we found that c-Myc was physically associated with p73␣ and significantly impaired the transcriptional activity of p73␣ on Bax and p21 waf1 promoters. Expression of MM1 strongly reduced the c-Myc-mediated inhibitory activity on p73␣. These results suggest that MM1 may act as a molecular partner for p73 to prevent the c-Mycmediated inhibitory effect on its activity.p73 is a new member of the p53 gene family (1). Like p53, p73 is a nuclear transcription factor, which carries an NH 2 -terminal transactivation domain, sequence-specific DNA-binding domain, and oligomerization domain. As expected from the significant amino acid sequence homology in the sequence-specific