Abstract
Negative regulation of the NF-κB transcription factor is essential for tissue homeostasis in response to stress and inflammation. NF-κB activity is regulated by a variety of biochemical mechanisms including phosphorylation, acetylation, and ubiquitination. In this study, we provide the first experimental evidence that NF-κB is regulated by SUMOylation, where the RelA subunit of NF-κB is SUMOylated by PIAS3, a member of the PIAS (protein inhibitor of activated STAT) protein family with E3 SUMO ligase activity. PIAS3-mediated NF-κB repression was compromised by either RelA mutant resistant to SUMOylation or PIAS3 mutant defective in SUMOylation. PIAS3-mediated SUMOylation of endogenous RelA was induced by NF-κB activation thus forming a negative regulatory loop. The SUMOylation of endogenous RelA was enhanced in IκBα null as compared with wild type fibroblasts. The RelA SUMOylation was induced by TNFα but not leptomycin B mediated RelA nuclear translocation. Furthermore, RelA mutants defective in DNA binding were not SUMOylated by PIAS3, suggesting that RelA DNA binding is a signal for PIAS3-mediated SUMOylation. These results support a novel negative feedback mechanism for NF-κB regulation by PIAS3-mediated RelA SUMOylation.
Highlights
NF-kB is a transcription factor that mediates cellular response to inflammation, immune response, and stress [1]
RelA SUMOylation by PIAS3 To determine whether RelA is SUMOylated by PIAS
To test whether endogenous RelA is SUMOylated by PIAS3, we evaluated RelA SUMOylation by in vivo SUMOylation assay in 3T3 cells transiently transfected with Flag-tagged PIAS and Histagged SUMO3
Summary
NF-kB is a transcription factor that mediates cellular response to inflammation, immune response, and stress [1]. Deregulation of NF-kB is one of the common features in many pathological disorders including inflammatory diseases and cancer. NF-kB is a dimeric protein which can be comprised of a variety of combinations of Rel family DNA binding proteins including RelA (p65), RelB, c-Rel, p50, and p52. A heterodimer of RelA and p50 is the most common combination in the canonical NFkB signaling pathway. NF-kB is held in check by the inhibitor of NF-kB (IkBa) which sequesters NF-kB in the cytoplasm and prevents NF-kB DNA binding. IkBa is phosphorylated by IkB kinases, leading to its degradation. The degradation of IkBa allows the free NF-kB to translocate to the nucleus where it functions as a transcription factor to induce the expression of proinflammatory cytokines, chemokines, and factors for cell proliferation and survival [2]
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