Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation

Abstract

In response to DNA damage, p73 plays a critical role in cell fate determination. In this study, we have found that Plk1 (pololike kinase 1) associates with p73, phosphorylates p73 at Thr-27, and thereby inhibits its pro-apoptotic activity. During cisplatinmediated apoptosis in COS7 cells in which the endogenous p53 is inactivated by SV40 large T antigen, p73 was induced to accumulate in association with a significant down-regulation of Plk1. Consistent with these observations, Plk1 reduced the stability of the endogenous p73. Immunoprecipitation and in vitro pulldown assay demonstrated that p73 binds to the kinase domain of Plk1 through its NH 2 -terminal region. Luciferase reporter assay and reverse transcription-PCR analysis revealed that Plk1 is able to block the p73-mediated transcriptional activation. Of note, kinase-deficient Plk1 mutant (Plk1(K82M)) retained an ability to interact with p73; however, it failed to inactivate the p73-mediated transcriptional activation, suggesting that kinase activity of Plk1 is required for the inhibition of p73. Indeed, in vitro kinase assay indicated that p73 is phosphorylated at Thr-27 by Plk1. Furthermore, small interference RNA-mediated knockdown of the endogenous Plk1 in p53-deficient H1299 cells resulted in a significant increase in the number of cells with sub-G 1 DNA content accompanied by the upregulation of p73 and pro-apoptotic p53 AIP1 as well as the proteolytic cleavage of poly(ADP-ribose) polymerase. Thus, our present results suggest that Plk1-mediated dysfunction of p73 is one of the novel molecular mechanisms to inhibit the p53-independent apoptosis, and the inhibition of Plk1 might provide an attractive therapeutic strategy for cancer treatment.p73 is one of newly identified p53 tumor suppressor gene family members (p53, p73, and p63) that encodes a nuclear transcription factor (1-3). Like the other p53 family members, p73 encodes multiple isoforms, including TA (transactivating), with distinct COOH-terminal extensions arising from alternative splicing events and ⌬N (nontransactivating) variants generated by alternative promoter usage (2-4). ⌬Np73 has an oncogenic potential (5) and displays a dominant-negative behavior toward TAp73 as well as wild-type p53 (6). TAp73 transactivates overlapping set of p53-target genes implicated in the induction of cell cycle arrest and/or apoptosis, and plays an important role in the regulation of DNA damage response, which is closely linked to its DNA binding activity. The initial studies demonstrated that TAp73 does not induce enough to accumulate in response to DNA damage arising from UV exposure or actinomycin D treatment (1); however, it has been shown that, in response to certain subset of DNA-damaging agents, TAp73 accumulates in the cell nucleus and exerts its pro-apoptotic function (7).Accumulating evidence suggests that TAp73 is regulated by post-translational modifications such as phosphorylation and acetylation. For example, TAp73 is stabilized in response to DNA damage such as cisplatin (CDDP) 2 treatment or e...

Plk1 regulates liver tumor cell death by phosphorylation of TAp63

“…On the other hand, KD Plk1(K82M) did not modify the protein stability of TAp63alpha. In addition, the Plk1-related degradation of TAp63 was not attenuated by a proteasome inhibitor MG132 (data not shown), consistent with the phenomenon in TAp73/Plk1 experiments (Koida et al, 2008). These results suggest that Plk1 downregulates the protein stability of TAp63 by its phosphorylation through a proteasome-independent pathway and suppresses TAp63-induced cell death.…”

“…Interestingly, the previously reported p63 phosphorylation was related to protein stabilization, upregulation of activity and induction of apoptotic cell death (Westfall et al, 2005;Suh et al, 2006). This study, however, clarified that phosphorylation by Plk1 results in protein degradation and suppression of the transcriptional activity of TAp63alpha (Figure 5b), consistent with the inactivation of transcriptional activity in p53 and p73alpha (Koida et al, 2008). It can be noted that, this Plk1-related TAp63 degradation was affected by cell density of Hep3B cells (data not shown), suggesting that further analysis of p63 phosphorylation and its effect on degradation will be required in future.…”

“…RNA extraction and RT-PCR Total RNA extraction, cDNA synthesis and semi-quantitative RT-PCR were carried out as described earlier (Koida et al, 2008). The specific primers were indicated as supplements (Supplementary data).…”

“…In fact, depletion of Plk1 by small interfering RNA (siRNA) treatment resulted in the arrest of cellcycle progression at the G2/M phase, such that proliferation was dramatically reduced and apoptosis increased in multiple cancer cell lines (Strebhardt and Ullrich, 2006); however, the downstream effectors and the molecular mechanism of Plk1-depletion-induced apoptosis have not been identified in detail. In the previous paper, we found that Plk1 inhibits the pro-apoptotic function of p53 through physical interaction and that Plk1 has the ability to bind to and phosphorylate transactivating (TA)p73 at Thr27, thereby inhibiting its transcriptional as well as pro-apoptotic activity (Koida et al, 2008), suggesting that p53 family molecules have a significant function in Plk1-depletion-induced apoptosis in cancer cells.…”

Enhanced chemosensitivity to CPT-11 in colorectal carcinoma xenografts by small hairpin RNA interference targeting PLK1

“…So far, the feasible approach is to explore Plk1 inhibitor blocking its function on the cell cycle progression or the antitumor drug target including small molecules, antisense oligonucleotides, small interference (si)RNA and so on [5][6][7]. It was reported that down-regulation of PLK1 expression or function could inhibit cell proliferation, advance apoptosis, sensitize the tumor cells to the antitumor agents and inhibit tumor growth in mice [8][9][10][11][12][13][14][15]. Wonderfully, RNA interference is an effective strategy for gene silencing that directly transfected into tumor cells to knock down endogenous gene expression [6].…”