Figure 5

PfNaps, histones and PfAlba are substrates for recombinant PfCK2α. In vitro kinase assays were performed on purified native parasite histones and recombinant PfNapL, PfNapS, four members of the PfAlba gene family, and Sir2, using active GST-PfCK2α and with a GST-PfCK2α K72M dead mutant. The PfCK2α kinase yielded an autophosphorylation band at 60 kDa, whereas the kinase-dead mutant did not yield any signal with any of the tested substrates. (A) GST-PfCK2α phosphorylates PfNapS and PfNapL, with PfNapL giving a much stronger signal. (Left and middle): active GST-PfCK2α; (right): kinase-dead GST-PfCK2α K78M mutant. (B) GST-PfCK2α specifically phosphorylated histone H2B (right panel, left lane) in a mixture of native histones purified from the parasite (left panel: Coomassie stain of the purified native histones used for the assay). The PfCK2α kinase-dead mutant did not yield any signal. (C) GST-PfCK2α phosphorylated PfAlba1 and PfAlba2 GST fusion proteins, producing signals at 53 and 51 kDa respectively (left panel, bottom bands). (Left panel) Active GST-PfCK2α; (right panel) kinase-dead GST-PfCK2α K78M mutant. (D) His-PfPK6 wass unable to phosphorylate PfNapS and PfNapL, but autophosphorylates and phosphorylates MBP. (E) GST-PfCK2α is unable to phosphorylate PfAlba3, PfAlba4, and Sir2 GST fusion proteins. Only the GST-PfCK2α autophosphorylation band was visible.