Figure 4

TgCdk7 is an active kinase which can phosphorylate TgRPB1-CTD.

(A) Coomassie gel showing purified recombinant GST-TgCyclinH, GST-TgCdk7, His-TgMat1, TgCdk2-His, His-TgRPB1-CTD and histone H1 proteins used in the kinase assays. (B) TgRPB1-CTD was phosphorylated (lane four) in the presence of TgCdk7 and TgCyclinH. The phosphorylation of TgRPB1-CTD was increased in the presence Mat1 (lane five). TgCdk7, TgCyclinH or TgMat1 alone failed to phosphorylate TgRPB1-CTD. Coomassie stained gel (lower panel) show loading of all the proteins and equal loading of TgRPB1-CTD. (C) Similarly, TgCdk7/TgCyclinH phosphorylated universal kinase substrate histone H1 and the effect was enhanced in the presence of Mat1. (D) Kinase assays were performed using TgCdk2, TgCyclinH and TgMat1 in the presence of histone H1 as a substrate. Weak phosphorylation of histone H1 was detected with alone TgCdk2. Robust phosphorylation of histone H1was observed in the presence of activated kinase Tg-Cdk2/CyclinH. The phosphorylation level of histone H1 remained unchanged in the presence of TgMat1. Coomassie stained gel (lower panel) show equal loading of histone H1. (E) Tg-Cdk7/CyclinH/Mat1 did not phosphorylate TgCdk2 (lane two and three) but phosphorylate TgRPB1-CTD (lane one). (F) TgCdk7 kinase activity showed progressive reduction in presence of increasing concentration of BS-181 in vitro. The CTD kinase activity was completely abolished at 40 nM concentration. Coomassie stained gel show equal loading of TgRPB1-CTD (lower panel). (G) TgCdk2 kinase activity was not inhibited at 40 nM concentration of BS-181. (H) Kinase assay using lysate prepared from RH parasites treated with Cdk7 inhibitor, BS-181 showed diminished phosphorylation of TgRPB1-CTD. (I) TgCdk7 immunodepleted parasite lysate showed decline in TgRPB1-CTD phosphorylation. Pre-immune sera depleted parasite lysate was used as control. Coomassie stained gel show equal loading of TgRPB1-CTD.