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

Peer reviewe

The Activation of IL-1-Induced Enhancers Depends on TAK1 Kinase Activity and NF-κB p65

The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells

A purified and reconstituted CDK6/cyclin complex phosphorylates NF-κB p65 Ser 536.

<p>(<b>A</b>) GST fusion proteins of cyclins D1 to D3, viral (v)-cyclin and CDK6 were expressed and purified in <i>E.coli</i>. Comparable amounts of GST, GST-cyclins and GST-CDK6 as judged from immunoblotting analysis (upper panel) were mixed with HeLa cell extract to provide CDK-activating kinase CAK as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Phelps1" target="_blank">[37]</a>. Kinase reactions were performed by addition of ATP. Then, GST alone or activated CDK6/cyclin complexes were purified by addition of GSH-beads, washed and used to phosphorylate the GST-p65_{354- 551} substrate <i>in vitro</i>. Phosphorylation of p65 Ser536 was detected by immunoblotting of kinase reaction mixtures (lower panel). Recombinant GST protein or kinase reactions without GST-CDK6/cyclin complexes (labeled no kinase) were used to determine background signals. (<b>B</b>) Increasing amounts of a recombinant CDK6/cyclin D1 complex purified to homogeneity from baculovirus-infected insect cells was mixed with GST-p65_{354- 551} and the kinase reaction was allowed to proceed over time. Phosphorylation of p65 at Ser536 was detected by immunoblotting. (<b>C</b>) HeLa cells were transfected with the expression vectors for wild type (wt) CDK6, a gain of function mutant (CDK6-S178P) and cyclin D3 or viral (v)- cyclin. 24 h later cells were lysed and phosphorylation of p65 and of Rb and expression of transfected proteins was analyzed by immunoblotting of cell extracts using the indicated antibodies.</p

Gene-specific regulation of NF-κB target genes by CDK6.

<p>(<b>A</b>) HEK293IL-1R cells were transiently transfected with a vector directing the synthesis of two different shRNA duplexes directed at CDK6 or an empty vector control along with a NF-κB-dependent reporter gene and the SV40-ß gal vector to allow for normalization. After 24 h, cells were stimulated for 4 h with IL-1α(10 ng/ml, black bars) or left untreated (white bars). Cells were lysed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. (<b>B</b>) HEK293IL-1R cells were transiently transfected with empty vector, NF-κB (3) luc and SV40-ß gal. After 24 h cells were pretretreated with 10 µM PD332991 and then further stimulated for 4 h with IL-1α(10 ng/ml) or left untreated. Shown is the mean luciferase activity +/− s.e.m. from two independent experiments. (<b>C</b>) HeLa Fucci cells were arrested for two days and then left untreated or were treated with 10 µM PD332991 (hatched bars) for 30 min followed by 30 min TNFα (20 ng/ml) as indicated. Thereafter, total RNA was prepared and analysed for the expression of the indicated mRNAs by RT-qPCR. Data show the mean –fold change +/− s.e.m. from two independent experiments performed in duplicate. The asterisk indicates significant differences (p< = 0.05) as determined by paired t-test.</p

Phosphorylation of Ser536 of p65 by CDK6/cyclin complexes contributes to activation of NF-κB-dependent transcription.

<p>(<b>A</b>) HEK293IL-1R cells were transiently transfected with the indicated amounts (µg) of expression vectors and a NF-κB-dependent luciferase gene. After 24 h cells were lyzed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. Expression of cotransfected MYC-cyclin D1, v-cyclin derived from Kaposi’s sarcoma herpesvirus (MYC-v-cyclin) and HA-CDK6 was analyzed by immunoblotting using anti MYC or anti CDK6 antibodies for all experiments in parallel. One representative blot is shown. (<b>B</b>) NF-κB p65-deficient MEFs stably reconstituted with wild type p65 (−/−/wt) or the Ser536A mutant (−/−/Ser536A) as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Okazaki1" target="_blank">[64]</a> were transiently transfected with empty vector or HA-CDK6 plus v-cyclin expression vectors. Cotransfected luciferase reporter genes were either controlled by three NF-κB binding sites (upper graph) or alternatively by the cyclin D1 promoter (lower graph). After 24 h cells were lyzed, luciferase activity was determined and normalized for co-transfected SV40-promoter driven ß-galactosidase activity. Shown is the mean luc. activity +/− s.e.m. from three (lower graph) or four (upper graph) independent experiments performed in duplicates relative to the vector control. The lower panel shows a representative immunoblot for the detection of p65 wt and Ser536Ala mutant, p65 Ser536 phosphorylation and ectopically expressed CDK6. Asterisks indicate significant changes of reporter gene activity.</p

p65 Ser536 phosphorylation is increased in v-cyclin expressing B-cell lymphomas, pre-tumorigenic lymphoid organs and further enhanced in thymic tumors.

<p>(<b>A</b>, <b>B</b>) Whole cell extracts were prepared from thymic (A) and splenic (B) lymphocytes isolated from non-transgenic littermates and mice expressing FLAG-v-cyclin under the control of the Eμ promoter which targets v-cyclin expression to B- and T-cell compartments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Verschuren2" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Verschuren3" target="_blank">[45]</a>. Lymphocytes from control thymi and spleens, pre-tumorigenic thymi and spleens, as well as thymic tumors were analysed by immunoblotting with antibodies against p65 phosphorylated at Ser536 (P-S536 p65), p65, CDK6, anti-FLAG (for v-cyclin) and cyclin D3. Anti-tubulin was used as loading control. Numbers indicate individual mice. (<b>C</b>) H&E staining of tissue sections corresponding to thymic tissues as described above.</p

CDK6 contributes to basal and TNF-inducible p65 Ser536 phosphorylation.

<p>(<b>A</b>) MDCK cells were treated for 24 h with siRNAs directed against CDK6 or adequate scrambled controls followed by treatment with TNFα as indicated. Total cell extracts were analyzed for phosphorylation of p65 and expression of the indicated proteins by immunoblotting. Numbers indicate relative levels of p65 Ser536 phosphorylation. (<b>B</b>) The experiment was done as in (A) with the exception that cells were fractionated in nuclear and cytosolic extracts. Extracts were further analyzed for the occurrence of nuclear p65, relative levels are indicated by numbers. Suppression of CDK6 in the cytoplasm was validated by CDK6 antibodies and quantified. Actin and HDAC1 detection served to control the purity of the fractions. (<b>C</b>) A Hela cell line stably transfected with shRNAs directed against CDK6 (shCDK6, K06) or cells stably transfected with pSuper-Puro (vector) were subjected to cell cycle arrest by serum deprivation for 48 h (arrest). Thereafter cells were released for 6 h by addition of 20% serum (G1 release). In addition, cells were treated with IL-1 (10 ng/ml) for 30 min as indicated. Total cell extracts were separated by SDS-PAGE in two sets on one gel and were transferred to one membrane. One half of the membrane was probed with antibodies against phospho-Ser536, CDK6, CDK4 and β-actin. The other half was probed with anti p65 antibodies. Numbers indicate relative phosphorylation of p65 or relative protein levels of p65 and CDK6. The graph shows the mean +/− s.e.m. of relative Ser536 phosphorylation normalized to p65 total protein as determined in three independent experiments. The asterisk indicates significant differences (p< = 0.018) as determined by paired t-test. (<b>D</b>) Naturally KSHV-infected primary effusion lymphoma (PEL) cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Arvanitakis1" target="_blank">[39]</a> were stably infected with lentiviruses encoding shRNAs directed against CDK6 or scrambled shRNA controls. Cells were fractionated into cytosolic (c), nuclear soluble (ns) and nuclear insoluble (ni) fractions. These extracts were analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control for cytosolic and nuclear soluble fractions and SP1 as a marker for the nuclear insoluble fraction. Numbers indicate relative levels of phospho-Ser536 p65, cyclinD3 and CDK6. (<b>E</b>) PEL cells silenced for CDK6 expression as described in (D) were analyzed by indirect immunofluorescence for p65 and p65 Ser536 phosphorylation with specific antibodies, nuclear DNA was revealed by DAPI staining.</p