Journal
CELLULAR SIGNALLING
Volume 26, Issue 7, Pages 1437-1444Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cellsig.2014.03.003
Keywords
GTPase; Rad; RelA/p65; NF kappa B; TNF alpha
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Funding
- Aim for the Top University Plan from the Ministry of Education, Taiwan [99AC-T512, 100AC-T506, 103AC-T307]
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Rad is a Ras-related small GTPase shown to inhibit cancer cell migration, and its expression is frequently lost in lung cancer cells. Here we provide evidence that Rad can negatively regulate the NF kappa B pathway. Overexpressing Rad in cells lowered both the basal and TNF alpha-stimulated transcriptional activity of NF kappa B. Compared with control cells, Rad-overexpressing cells displayed more cytoplasmic distribution of the NF kappa B subunit RelA/p65, while Rad-knockdown cells had higher levels of nuclear RelA/p65. Depleting Rad did not affect the kinetics of TNF alpha-induced I kappa B degradation, suggesting that Rad-mediated regulation of NF kappa B was through an I kappa B-independent mechanism. Expression of a nucleus-localized mutant Rad was sufficient to inhibit the NF kappa B transcriptional activity, whereas expressing the scaffolding protein 14-3-3 gamma to retain Rad in the cytoplasm alleviated the suppressive effect of Rad on NF kappa B. GST pull-down assays showed that Rad could directly bind to RelA/p65, and co-immunoprecipitation demonstrated that the Rad-p65 interaction primarily occurred in the nucleus. Adding Rad-containing nuclear extracts or purified GST-Rad in the electrophoretic mobility shift assays dose-dependently decreased the binding of RelA/p65 to an oligonucleotide probe containing the NF kappa B response element, suggesting that Rad may directly impede the interaction between RelA/p65 and DNA. Rad depletion altered the expression of an array of NF kappa B target genes, including upregulating MMP9. Knockdown of Rad expression in cells increased both basal and TNF alpha-stimulated MMP9 activities and cell invasion. Collectively, our results disclose a novel role of nuclear Rad in inhibiting the NF kappa B pathway function. (C) 2014 Elsevier Inc. All rights reserved.
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