NF-κB RelA phosphorylation regulates RelA acetylation

The nuclear functions of NF-kappa B p50/RelA heterodimers are regulated in part by posttranslational modifications of its ReIA subunit, including phosphorylation and acetylation. Acetylation at lysines 218, 221, and 310 differentially regulates RelA's DNA binding activity, assembly with I kappa B alpha, and transcriptional activity. However, it remains unclear whether the acetylation is regulated or simply due to stimulus-coupled nuclear translocation of NF-kappa B. Using anti-acetylated lysine 310 ReIA antibodies, we detected p300-mediated acetylation of ReIA in vitro and in vivo after stimulation of cells with tumor necrosis factor alpha (TNF-alpha). Coexpression of catalytically inactive mutants of the catalytic subunit of protein kinase A/mitogen- and stress-activated kinase 1 or IKK1/IKK2, which phosphorylate ReIA on serine 276 or serine 536, respectively, sharply inhibited ReIA acetylation on lysine 310. Furthermore, phosphorylation of ReIA on serine 276 or serine 536 increased assembly of phospho-ReIA with p300, which enhanced acetylation on lysine 310. Reconstitution of ReIA-deficient murine embryonic fibroblasts with ReIA S276A or ReIA S536A decreased TNF-alpha-induced acetylation of lysine 310 and expression of the endogenous NF-kappa B-responsive E-selectin gene. These findings indicate that the acetylation of ReIA at lysine 310 is importantly regulated by prior phosphorylation of serines 276 and 536. Such phosphorylated and acetylated forms of ReIA display enhanced transcriptional activity.