Research ArticleImmunology

Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

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Science Signaling  23 Aug 2016:
Vol. 9, Issue 442, pp. ra85
DOI: 10.1126/scisignal.aab2820

IKK is the start and stop signal

By phosphorylating inhibitor of κB (IκB), the kinase IKK targets it for destruction, thus enabling the dimeric transcription factor nuclear factor κB (NF-κB) to translocate to the nucleus and alter target gene expression. Pradère et al. generated knock-in mice expressing a mutant p65 subunit of NF-κB that could not be phosphorylated by IKK at Ser534 (the mouse homolog of human p65 Ser536). The mutated p65 translocated to the nucleus properly, but the protein exhibited enhanced stability, which resulted in exacerbated responses to inflammatory stimuli in vivo. Experiments with human cells indicated that this regulatory mechanism was conserved. Thus, in addition to stimulating NF-κB signaling by phosphorylating IκB, IKK limits inflammation by targeting this regulatory site in mouse and human p65.


Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of κB (IκB) kinase (IKK)–dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. We investigated the contribution of the phosphorylation of the NF-κB subunit p65 at the IKK phosphorylation site serine 536 (Ser536) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser536), we observed increased expression of NF-κB–dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κB signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser534-phosphorylated wild-type protein. Together, our results suggest that Ser534 phosphorylation of p65 in mice (and, by extension, Ser536 phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κB signaling to prevent deleterious inflammation.

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