Phosphorylation of Serine 536 of p65(RelA) Downregulates Inflammatory Responses.

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammatory diseases and immune responses. Recently, a novel transcriptional regulatory mechanism of NF-κB involving the phosphorylation of serine 536 (534 in mice; S534) of its p65 subunit was reported; however, further research is required to elucidate the physiological role of S534 phosphorylation. Therefore, we generated S534A knock-in (KI) mice, in which the S534 of p65 was substituted with alanine. Similar to the wild-type (WT) mice, S534A KI mice developed normally. After stimulation with tumor necrosis factor α (TNFα), mouse embryonic fibroblasts (MEFs) derived from S534A KI mice exhibited increased target gene expression compared with that in the WT MEFs, which was induced by long-term binding of p65 to DNA. According to comprehensive gene expression analysis after stimulation with TNFα, the expression of genes p65ted to inflammatory and immune responses was increased, and the expression of genes p65ted to lipolysis was decreased in S534A KI MEFs. Analyses of a periodontal disease model established using WT and S534A KI mice revealed that alveolar bone resorption was enhanced in S534A KI mice owing to an increase in the number of osteoclasts, which was not attributed to the differentiation of osteoclast precursor cells but to an increased expression of interleukin-1β and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.

Aoki T ，Gao J ，Li A ，Huang F ，Tu Y ，Wu W ，Matsuda M ，Kiyoshima T ，Nishimura F ，Jimi E ... -  《-》

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

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 (Ser(536)) 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 Ser(536)), 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 Ser(534)-phosphorylated wild-type protein. Together, our results suggest that Ser(534) phosphorylation of p65 in mice (and, by extension, Ser(536) phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κB signaling to prevent deleterious inflammation.

Pradère JP ，Hernandez C ，Koppe C ，Friedman RA ，Luedde T ，Schwabe RF ... -  《-》

A novel NF-kappaB pathway involving IKKbeta and p65/RelA Ser-536 phosphorylation results in p53 Inhibition in the absence of NF-kappaB transcriptional activity.

Jeong SJ ，Pise-Masison CA ，Radonovich MF ，Park HU ，Brady JN ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II.

Wang D ，Westerheide SD ，Hanson JL ，Baldwin AS Jr ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Activation of nuclear factor-kappaB-dependent transcription by tumor necrosis factor-alpha is mediated through phosphorylation of RelA/p65 on serine 529.

Wang D ，Baldwin AS Jr 《JOURNAL OF BIOLOGICAL CHEMISTRY》