Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

Zhongkui Li,Jiangong Niu,Tadashi Uwagawa,Bailu Peng,Paul J Chiao

doi:10.1074/jbc.m410119200

Abstract

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

Highlights

RelA, the p65 subunit of the NF-␬B transcription factor, plays a key role in protecting cells from proapoptotic stimuli [1,2,3]
Small interfering RNA-mediated inhibition of polo-like kinase 3 (Plk3) expression suggests that Plk3 is essential for superoxide-induced cell death, and deletion analysis of Plk3 showed that the N-terminal domain is essential for induction of delayed onset of apoptosis
Plk3 Expression Is Induced by Various Stimuli—To determine whether Plk3 expression is induced by other NF-␬B inducers in different cells, we stimulated HEK293/Puro and HEK293/I␬B␣M cells with doxycycline (50 ␮g/ml), MDAPanc28/Puro, and MDAPanc-28/I␬B␣M with TNF-␣ (10 ng/ml) and NIH3T3 cells with fibroblast growth factor-1 (20 ng/ml) and phorbol 12-myristate 13-acetate (30 ␮g/ml) for various times, as indicated, and isolated total RNA for measuring the levels of

Summary

Introduction

RelA, the p65 subunit of the NF-␬B transcription factor, plays a key role in protecting cells from proapoptotic stimuli [1,2,3]. In the same cells in which inhibition of NF-␬B promotes the induction of apoptosis by glucocorticoids, NF-␬B is required for the induction of apoptosis by stimulation of phorbol ester and ionomycin for mimicking T-cell activation [12] These findings suggest that whether the function of NF-␬B is proapoptotic or antiapoptotic in a given cell depends on the cell type, extent of NF-␬B activation, and nature of the apoptotic signals. Small interfering RNA (siRNA)-mediated inhibition of Plk expression suggests that Plk is essential for superoxide-induced cell death, and deletion analysis of Plk showed that the N-terminal domain (amino acids 1–26) is essential for induction of delayed onset of apoptosis. Our results suggest that Plk is an NF-␬B-regulated kinase that mediates p53-dependent and -independent proapoptotic response in reaction to elevated levels of superoxide

Methods

Results

Conclusion