Identification of CDK1 as a direct H2B S6 kinase. (A) Flag-tagged CDK1 forms were expressed with cyclin B1-Venus in 293T cells. CDK1 was immunoprecipitated with anti-Flag M2 affinity gel and cyclin B1 with GFP-Trap beads for a subsequent in vitro kinase assay with recombinant H2B. Samples were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) 293T cells were transfected to express cyclin B1-Venus or GFP as shown. Cells were lysed with NP-40 buffer and GFP, and cyclin B1-Venus were immunoprecipitated with the GFP-Trap for a subsequent in vitro kinase assay using recombinant H2B as a substrate protein. The reactions were incubated in the presence or absence of 1 µg recombinant p21CIP1 protein (Sigma) as shown, H2B S6ph was analyzed by immunoblotting. (C) 1 µg of recombinant and purified CDK1-cyclin B1 (Thermo Fisher) or 2 µg GST control (Sigma) were incubated with recombinant H2B (2 µg) in the presence of ATP. The reaction was analyzed for histone phosphorylation by Western blotting (top) and protein integrity by SDS-PAGE and CBB staining. (D) U2OS F4 2B8 cells with an array of lacO sites integrated close to the centromere of chromosome 2 were transfected with GFP-lacI alone or a nondegradable form of cyclin B1 (cyclin ND) fused to GFP-lacI and Flag-tagged forms of CDK1 AF and CDK1 KD. After 24 h, cells expressing moderate amounts of the GFP-lacI fusion proteins were fixed for immunofluorescence and analyzed for phosphorylation of H2B S6 and the H3 S10 control as well as for Flag-CDK1 localization. The left part shows representative results from the immunofluorescence studies. The right part displays the ratio of cells showing colocalization of GFP-lacI-cyclin B1 and H2B S6ph, Flag-CDK1, or H3 S10ph stained with the indicated antibodies. n > 100 cells from two independent experiments were analyzed for each condition; error bars show standard deviations between the two biological replicates. Bars, 5 µm.
