No abstract

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 phosphorylation, 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 d...

The activity of NF-B is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-B and the functional role of these phosphorylations are still being uncovered.

NF-kappaB is a generic name for an evolutionarily conserved transcription-factor system that contributes to the mounting of an effective immune response but is also involved in the regulation of cell proliferation, development, and apoptosis. The implication of NF-kappaB in central biological processes and its extraordinary connectivity to other signaling pathways raise a need for highly controlled regulation of NF-kappaB activity at several levels. While all NF-kappaB activation pathways share a central and critical proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of DNA-binding subunits, there is evidence for a downstream level of NF-kappaB regulation that employs several mechanisms. These include promoter-specific exchange of dimers and modification of the transactivating p65 subunit by phosphorylation, acetylation, ubiquitination, or prolyl isomerization. The signaling pathways and enzymes controlling this second level of regulation and their potential use as therapeutic targets for the treatment of NF-kappaB-associated pathologies are discussed here.

Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.