Activation of Polo-like kinase 3 by hypoxic stresses

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.

FIGURE 1.

Hypoxia/reoxygenation-induced activation of Plk3 and c-Jun phosphorylation in HCE Cells. A, effect of hypoxia (Hypo) and reoxygenation (Reox) on Plk3 activity. Immunocomplex kinase assays were performed to determine Plk3 kinase activity, and hypophosphorylated GST-c-Jun fusion protein was used as a substrate. B, effect of hypoxia-activated Plk3 on c-Jun phosphorylation after removing JNK. JNK protein was removed from cell lysates by immunoprecipitation, and Plk3 activity was measured by immunocomplex kinase assay. C, hypoxia/reoxygenation-induced Plk3 activation and c-Jun phosphorylation. Western blots were used to detect Plk3 expression and phosphorylated c-Jun using antibody specific to phospho-c-Jun. D, dose-dependent response of CoCl₂-induced phosphorylation of c-Jun. E, time course of hypoxia-induced site-specific phosphorylation of c-Jun at Ser-63 and Ser-73. F, effect of hypoxia and reoxygenation on AP-1 DNA binding activity detected by EMSA and supershift. UV irradiation-induced phosphorylation of c-Jun and AP-1 activation were detected as positive controls.

FIGURE 2.

Hypoxia/reoxygenation-induced activation of MAP kinases. Effect of hypoxia (Hypo) and reoxygenation (Reox) on: A, JNK phosphorylation; B, SEK phosphorylation; C, Erk phosphorylation; and D, p38 phosphorylation. UV irradiation-induced MAP kinase activation served as positive controls. Activation of SEK and MAP kinases were determined by detecting hypoxia-induced changes in phosphorylation levels of these proteins with specific anti-phospho-antibodies. Statistical significance was indicated by symbols of * and ** for p < 0.05 and p < 0.01, respectively.

FIGURE 3.

Immunocolocalization of Plk3 and c-Jun. A, co-localization of Plk3 and c-Jun in hypoxia-induced cells. B, co-localization of Plk3 and c-Jun in nuclei. Cellular Plk3 and c-Jun proteins in control and hypoxia-induced HCE cell were stained using monoclonal antibodies specific to Plk3 and c-Jun, respectively. Cell nuclei were stained by DAPI.

FIGURE 4.

Effect of Plk3 mutants on c-Jun phosphorylation and AP-1 activation. A, effect of overexpressing wild-type Plk3 and kinase-defective Plk3 mutant (Plk3^K52R) on c-Jun phosphorylation. B, effect of overexpressing constitutively active Plk3 (Plk3-PBD) and defective Plk3 (Plk3-WV) on c-Jun phosphorylation. C, effect of overexpressing constitutively active Plk3 (Plk3-PBD) and defective Plk3 (Plk3-WV) on AP-1 activation detected by EMSA. Control experiments were performed by transfecting HCE cells with GFP-tagged Plk3 and the vector only. D, effect of knocking-down Plk3 using siRNA on hypoxia-induced c-Jun phosphorylation. UV irradiation-induced c-Jun phosphorylation served as positive controls. The symbol * indicates a significant difference (p < 0.05, n = 3).

FIGURE 5.

Effect of Plk3 on hypoxia-induced apoptosis. A, detection of hypoxia-induced DNA fragmentation. B, activation of caspase 3 induced by hypoxia and reoxygenation. UV irradiation-induced caspase 3 activation served as the positive control. C, effect of overexpressing constitutively active Plk3 (Plk3-PBD) and defective Plk3 (Plk3-WV) on caspase 3 activation. The symbol * indicates a significant difference (p < 0.01, n = 4).

FIGURE 6.

Effect of Plk3 on hypoxia/reoxygenation-induced changes in survival cell index detected by MTT assay. A, hypoxia-induced decrease in survival cell index. B, CoCl₂ induced a decrease in survival cell index. C, effect of overexpressing wild-type Plk3 and kinase-defective Plk3 mutant (Plk3^K52R) on survival cell index. D, effect of overexpressing constitutively active Plk3 (Plk3-PBD) and defective Plk3 (Plk3-WV) on hypoxia-induced survival cell index. The symbol * indicates a significant difference (p < 0.05, n = 4).