Nuclear factor kappa-B (NF-kB) 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-kB 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-kB activation was revealed upon RNAimediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-kB and TNFinduced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-kB 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-kB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-kB to chronic inflammation and neoplasia. Citation: Buss H, Handschick K, Jurrmann N, Pekkonen P, Beuerlein K, et al. (2012) Cyclin-Dependent Kinase 6 Phosphorylates NF-kB P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression. PLoS ONE 7(12): e51847. doi:10.1371/journal.pone.0051847 Editor: Joao P.B. Viola, National Cancer Institute (INCA), Brazil Received May 29, 2012; Accepted November 7, 2012; Published December 26, 2012 Copyright: 2012 Buss et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft Kr1143/7-1, TRR81 (B2), Na292/9-1, SFB854 (TP4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Naumann@med.ovgu.de (MN); Michael.Kracht@pharma.med.uni-giessen.de (MK) . These authors contributed equally to this work.