Nucleolar Sequestration of RelA (p65) Regulates NF-κB-Driven Transcription and Apoptosis

Abstract

The molecular mechanisms that regulate nuclear NF-κB to determine whether the stimulation of this pathway has a pro- or antiapoptotic effect on cells have yet to be fully defined. Nuclear compartmentalization is increasingly recognized as an important mechanism for regulating the activity of transcription-related proteins and modulating cell growth and death. We have investigated whether such compartmentalization serves as a mechanism for regulating NF-κB transcriptional activity. We demonstrate that the RelA component of NF-κB is sequestered in the nucleolus in response to the proapoptotic NF-κB stimuli aspirin, serum withdrawal, and UV-C radiation. In contrast, RelA is excluded from the nucleolus in response to the cytokines tumor necrosis factor and TRAIL. We identify an N-terminal motif of RelA that is essential for the nucleolar localization of the protein and show that deleting this motif inhibits the translocation of RelA from the nucleoplasm to the nucleolus. We demonstrate that the nucleolar accumulation of RelA is paralleled by a decrease in basal levels of NF-κB transcriptional activity and by apoptosis. Furthermore, we show that the retention of RelA in the nucleoplasm inhibits this decrease in NF-κB-driven transcription and blocks apoptosis induced by aspirin and UV-C radiation. This work identifies a novel cellular mechanism for regulating NF-κB-driven transcription and apoptosis, involving the nucleolar sequestration of a key NF-κB subunit. These data contribute to the understanding of the complexities of NF-κB function and have considerable relevance to cancer prevention and therapy.

ACKNOWLEDGMENTS

We gratefully acknowledge the gifts from Ron T. Hay (University of St. Andrews) of the IκBα antibody, the HIV-LTR and 3enhancer CON-A luciferase reporter constructs, and the rela null mouse embryo fibroblasts, and we thank him for critically reviewing this report. We also thank E. Quarnstrom (University of Sheffield) for kindly providing the RelA-GFP expression vector. P. Perry wrote the scripts for image capture, K. Reid was responsible for the isolation of nucleoli, and A. Sanderson provided help with cell sorting.

The work was supported by grants from the Scottish Executive Chief Scientist's Office (K/MRS/50/C2719 and CZB/4/41) and the AICR (02-330). L.A.S. is a Caledonian Research Fellow. M.G.D. is supported by Cancer Research United Kingdom Programme Grant funding (C348/A3758).