Abstract
In response to DNA damage, p73 plays a critical role in cell fate determination. In this study, we have found that Plk1 (polo-like kinase 1) associates with p73, phosphorylates p73 at Thr-27, and thereby inhibits its pro-apoptotic activity. During cisplatin-mediated 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 up-regulation 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.
Highlights
P73 is one of newly identified p53 tumor suppressor gene family members (p53, p73, and p63) that encodes a nuclear transcription factor [1,2,3]
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]; 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]
Summary
Cell Lines and Transfection—African green monkey kidney COS7, human osteosarcoma SAOS-2, U2OS, and human cervical carcinoma HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 g/ml penicillin, and 50 g/ml streptomycin (Invitrogen). COS7 and H1299 cells were transfected with the indicated combinations of the expression plasmids using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were washed in PBS, fixed in 3.7% formaldehyde for 1 h at room temperature, and permeabilized with 0.1% Triton X-100 for 5 min on ice. The cell nuclei were stained by DAPI. The purified Plk was added to the reaction mixture containing 50 M ATP and polyclonal anti-phospho-Thr antibody and incubated for 30 min at room temperature. GST or the indicated GST-p73␣ deletion mutants dissolved in substrate solution were added into the reaction mixture and incubated for 5 min at room temperature, followed by the measurement of absorbance in each well using a spectrophotometric. Total RNA and whole cell lysates were prepared 48 h after transfection
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