Plk1 inhibites the kinase activity of the P-TEFb complex.

<p>(A) <i>In vitro</i> kinase assay. HCT116 cells transfected with pCMV FLAG-cyclin T1 were synchronized into different phases as described in Material and Method. The synchronization of the cells was detected by FACS. Cell lysates were immunoprecipated with FLAG antibody. Half of the immunoprecipates were subjected to immunoblotting with Cdk9 and FLAG antibody. The other half of the immunoprecipates were then incubated with GST-RNA Pol II CTD in the presence of [γ-³²P] ATP for the <i>in vitro</i> kinase assay. (B) FLAG-cyclin T1 over-expressed in 293T cells were immunoprecipated with FLAG antibody. The immunoprecipitated cyclin T1 complexes were preincubated with the lysates from HeLa cells either asynchronized or synchronized in M phase with cold ATP for the <i>in vitro</i> kinase assay, and washed with kinase buffer. Then the FLAG-cyclin T1 complexes were incubated with GST-RNA Pol II CTD and [γ-³²P] ATP for a second round of <i>in vitro</i> kinase assay. The expression level of Plk1, phosphorylation of histone H3 in HeLa cells and the immunoprecipitated FLAG-cyclin T1 after incubating with Hela cell extracts and washed were detected by immunoblotting with the indicated antibody. (C) 293T cells were transfected with pCMV FLAG-cyclin T1 or pCMV FLAG-Cdk9 respectively. The cell lysates were immunoprecipitated with FLAG antibody and the immunoprecipitated complexes were subjected to <i>in vitro</i> kinase assay in the presence or absence of His-Plk1 TD or His-Plk1 KD with GST- RNA Pol II CTD as the substrate. (D) pCMV FLAG-cyclin T1 transfected 293T cells were treated with or without BI2536(1µM) for 3.5 h before harvest. The FLAG-cyclin T1 complexes were immunoprecipated with FLAG antibody and subjected to <i>in vitro</i> kinase assay with GST-RNA Pol II CTD as the substrate. Equal amount of the immunoprecipated FLAG-cyclin T1 and Cdk9 was shown. (E) FLAG-tagged cyclin T1, cyclin T1 S564A, cyclin T1 S564D over-expressed in 293T cells were immunoprecipitated with FLAG antibody and incubated with GST- RNA Pol II CTD and [γ-³²P] ATP for the <i>in vitro</i> kinase assay. The immunoprecipated FLAG-cyclin T1 and Cdk9 were subjected to immunoblotting.