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.