Cyclin-Dependent Kinase 6 Phosphorylates NF-κB P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression

Holger Buss,Pirita Pekkonen,Michael Naumann,Michael Kracht,Päivi M Ojala,Robin Wait,Knut Beuerlein,M Lienhard Schmitz,Jeremy Saklatvala,Helmut Müller,Nadine Jurrmann,Katja Handschick,Joao Pb Viola

doi:10.1371/journal.pone.0051847

Abstract

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.

Highlights

The transcription factor nuclear factor kappa B (NF-kB) comprises homo-or heterodimeric combinations of five DNAbinding subunits which regulate the expression of a large number of genes in multiple physiological or pathophysiological conditions such as inflammation, immune reactions or cancer [1]
While the p65 Ser536 phosphorylating kinases IKKa, IKKb, TBK1 and IKKe all eluted at higher NaCl concentrations, an unknown Ser536-specific enzymatic activity eluted from the column very early [4]
Since there are only a few well established substrates for Cyclin-dependent kinase 6 (CDK6) such as retinoblastoma protein (Rb) [22,24], histone H1 [30], Bcl-2 [31], Runx [32] and nucleophosmin [33] we performed additional in vitro experiments with the purified fractions to confirm that CDK6 is a direct p65 NF-kB kinase

Summary

Introduction

The transcription factor nuclear factor kappa B (NF-kB) comprises homo-or heterodimeric combinations of five DNAbinding subunits which regulate the expression of a large number of genes in multiple physiological or pathophysiological conditions such as inflammation, immune reactions or cancer [1]. Phosphorylation-dependent proteolytic degradation of IkBs in response to inducers such as proinflammatory cytokines, in particular IL-1 or TNFa, is followed by nuclear translocation and DNA binding of NF-kB subunits. There is an additional layer of regulation of NF-kB activity provided by numerous posttranslational modifications (PTMs) including ubiquitination, acetylation and phosphorylation [3]. According to the NF-kB barcode hypothesis that was recently suggested by us the differential modifications of the DNA-binding subunits generate distinct patterns that function to direct transcription in a highly target gene-specific fashion [11]

Methods

Results

Conclusion