Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway.

Suqing Xie,Meena Jhanwar-Uniyal,Peter Stambrook,Qi Wang,Intisar Husain,Huiyun Wu,John P Cogswell,Chris Conn,Wei Dai

doi:10.1074/jbc.m106050200

Abstract

Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997) J. Biol. Chem. 272, 28646-28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029-6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3(K52R)) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

Highlights

In mammals, DNA damage induced by ionizing radiation or UV light is detected by two serine/threonine kinases known as “mutated in ataxia telangiectasia” (ATM)1 and “ATM-related” (ATR) [3,4,5]
A model is proposed that explains the mechanism of action of Plk3 during genotoxic stress-induced activation of the DNA damage checkpoint, which results in cell cycle arrest and/or apoptosis
Given that the kinase activity of Plk1 has been shown to be down-regulated during activation of the DNA damage checkpoint [18] and that the antibody to Plk3 used for our immunocomplex kinase assay did not cross-react with human Plk1 (Fig. 2C), we measured the kinase activities of both Plk1 and Plk3 in the same A549 cells treated with adriamycin

Summary

Introduction

DNA damage induced by ionizing radiation or UV light is detected by two serine/threonine kinases known as “mutated in ataxia telangiectasia” (ATM)1 and “ATM-related” (ATR) [3,4,5]. We have provided evidence indicating that Plk3 is involved in DNA damage checkpoint response and that it may target p53 in vivo through regulation of phosphorylation on serine 20.

Results

Conclusion