Figure 5
From: MTBP phosphorylation controls DNA replication origin firing
MTBP is phosphorylated by cell cycle CDK and Cdk8/19-cyclin C. (A) Phosphorylation-mediated gel shifts of MTBP and Treslin/TICRR in mitotic cells depend on M-CDK. Cells arrested in mitosis with nocodazole (Noc) or asynchronous cells were treated for 30 min with DMSO, low (9 µM) or high concentrations (90 µM) of RO-3306 (RO.), roscovitine (Rosc.) or senexin A (Sen.). After cell lysis lysates were treated with lambda phosphatase (PPase) where indicated before immunoblotting using antibodies against MTBP (12H7) and Treslin/TICRR (148). (B) In vitro phosphorylation of MTBP by S-CDK. Recombinant 6His-Treslin/TICRR-aa1-1258-MTBP-Strep was incubated with Cdk2-cyclin A in the presence of γ‐32P‐ATP and DMSO or CDK inhibitor roscovitine and detected by autoradiography. Coomassie staining controlled loading (Load.). Rec., recombinant. (C) S-CDK phosphorylation of MTBP depends on CDK consensus sites. C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-6A or control plasmid were transiently transfected into 293T cells before anti-GFP IP, incubation with buffer or Cdk2-Cyclin A in the presence of γ‐32P‐ATP, and detection by autoradiography and immunoblotting using an antibody against MTBP (12H7). (i), autoradiogram; (ii), quantification of signal intensities of two independent replicates. WT signals were assigned the value 1 and control plasmid 0. MTBP-6A, amino acid exchanges S539A, T635A, S639A, S703A, S707A, T799A. (D,E) MTBP phosphorylation depends on binding to Cdk8/19-cyclin C. Native lysates of cells transfected as in C were immunoprecipitated and incubation with γ‐32P‐ATP before detection by autoradiography or immunoblotting using an antibody against MTBP (12H7) and Cdk8. (F) Native lysates of cells transfected with the indicated MTBP versions and with Cdk8-cyclin C were analysis as in D in the presence of DMSO or Cdk8 inhibitor senexin A. (i), autoradiogram; (ii), quantification of signal intensities of three independent replicates. WT signal intensities were assigned the value 1 and Cdk8 inhibitor treatment 0.
