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
Holger Buss, Anneke Dörrie, M. Lienhard Schmitz, Elke Hoffmann, Klaus Resch, Michael Kracht
2004
Journal of Biological Chemistry
Phosphorylation of NF-B 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
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... ion, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IB kinase (IKK)-␣/ (the best characterized Ser-536 kinases so far) or the IKK 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 IKK␣/-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 IKK␣, IKK, IKK⑀, and TBK1. Overexpressed IKK⑀ 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. The transcription factor NF-B regulates the expression of a large number of genes with important functions in the immune response, inflammation, cellular stress reactions, carcinogenesis, and apoptosis. In resting cells NF-B is trapped in the cytoplasm by its interaction with the inhibitor IB. A central step in activation of NF-B is the stimulus-induced phosphorylation of IB by IB kinases (IKK) 1 ␣ and . Both IKKs, IB, and NF-B subunits form a large signaling complex (1). Phosphorylation of IB results in targeting of IB to the proteasome followed by release and nuclear translocation of NF-B. Recent evidence suggests that NF-B activity is determined by additional mechanisms. Cells lacking the protein kinases glycogen synthase kinase 3 (2), TBK1/NF-kB-activating kinase (3, 4), IKK⑀ (5), NF-kB-inducing kinase (6), and protein kinase C (7) show a normal IB degradation pathway but impaired activation of NF-B-dependent gene expression. Furthermore, biochemical and genetic experiments in cells deficient for IKK␣ or IKK strongly suggest that direct phosphorylation and other modifications of NF-B are essential for NF-B function (8 -10). The complexity of this regulation is exemplified by recent studies that identified four serine residues in the p65 NF-B subunit that are inducibly phosphorylated by TNF or LPS and that are targeted by distinct signaling pathways. Phosphorylation of serine 276 of p65 occurs by catalytic subunit of protein kinase A in response to LPS (11, 12) or by mitogen-and stress-activated protein kinase in response to TNF (13). Phosphorylation at Ser-276 promotes its interaction with the coactivator CREB-binding protein (a histone acetylase) and is required for TNF-induced IL-6 expression (14, 15). Phosphorylation in the REL homology domain of p65 at Ser-311 is required for efficient p65 transactivation mediated by protein kinase C (7, 16, 17).
doi:10.1074/jbc.m409825200
pmid:15489227
fatcat:uspwscmoxbcopftmz3f6wyz3j4
