Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-κB at Serine 536 Is Mediated by Multiple Protein Kinases Including IκB Kinase (IKK)-α, IKKβ, IKKϵ, TRAF Family Member-associated (TANK)-binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATA-binding Protein-associated Factor II31-mediated Interleukin-8 Transcription

• Published in: The Journal of biological chemistry 2004-12, Vol.279 (53), p.55633-55643
• Main Authors: Buss, Holger, Dörrie, Anneke, Schmitz, M. Lienhard, Hoffmann, Elke, Resch, Klaus, Kracht, Michael
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409825200

Phosphorylation of NF-κB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IκB kinase (IKK)-α/β (the best characterized Ser-536 kinases so far) or the IKKβ inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKα/β-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKα, IKKβ, IKKϵ, and TBK1. Overexpressed IKKϵ and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter.