Activation of Polo-like Kinase 3 by Hypoxic Stresses

Ling Wang,Jie Gao,Wei Dai,Luo Lu

doi:10.1074/jbc.m801326200

Abstract

Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl(2). Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells.

Highlights

Signaling events and gene expression that control important cellular function affecting cell cycle progression and apoptosis
For Plk[3] kinase of hypoxia-induced Plk[3] activation on c-Jun phosphorylation assays, the experiments were carried out by incubation of the was further determined by two-step immunocomplex kinase immunocomplex of Plk[3] with GST-c-Jun fusion protein in 30 assays
␮l of kinase buffer (20 mM HEPES pH 7.6, 20 mM MgCl2, 25 mM immunoprecipitation, and Plk[3] was further immunopreglycerophosphate, 1 mM sodium orthovanadate, 2 mM dithio- cipitated from the cell extracts in that Jun N-terminal kinase (JNK)-1 protein had been threitol, 20 ␮M ATP, and, 10 ␮Ci of [32P]ATP) for 30 min at depleted

Summary

Introduction

Signaling events and gene expression that control important cellular function affecting cell cycle progression and apoptosis. Plk[3] and c-Jun Phosphorylation—We investigated hypoxic stress-induced signaling events in hypoxia and reoxygenation-exposed HCE cells and found that hypoxic stress including hypoxia and reoxygenation elicited increased Plk[3] kinase activities by detection of increased phosphorylation of c-Jun fusion protein in immunocomplex kinase assays (Fig. 1A). We investigated whether hypoxic stress induced c-Jun activation through the JNK signaling pathway in corneal epithelial cells.

Results

Conclusion