Figure 2
Both T232 and T236 of human Aurora B are important for its kinase activity in vitro. (a) Mutations introduced at T232 and T236 in human Aurora B. The mutants of the Aurora B were expressed as C-terminally Flag-tagged forms72 in 293 T cells and purified by using Flag-antibody beads. (b) Flag-tagged human Aurora B, as indicated, and Flag-tagged INCENP (IN-box polypeptide; aa803-918) proteins were separately purified from 293 T cells. The purified Aurora B was incubated with HH3 in the presence or absence of INCENP. WT, wild-type; KD, kinase-dead (D200N; D200 of the conserved HRD was changed to N). (c) The 293 T extracts containing HA-tagged INCENP (IN-box polypeptide) and purified Flag-tagged Aurora B mutants, as indicated, were mixed and immunoprecipitated with anti-Flag antibody, followed by kinase assays in the presence of HH3 and ATP. The presence of HA-INCENP in the immunoprecipitates was confirmed by western blotting using anti-HA antibody. Phosphorylation was detected by HH3 pS28 antibody. (d) The wild-type and mutant Aurora B proteins, as indicated, and INCENP were incubated in the presence or absence of Cdc7-ASK. In (b,c,d), phosphorylations were detected by western analyses using pT210 and HH3 pS28 antibodies. (e) Human Aurora B KD (D200N) was further mutated at Thr232 and Thr236. These human Aurora B (60 ng) mutants were incubated in in vitro kinase assays with [γ-32P] ATP in the absence or presence of Cdc7-ASK (25 ng). (f) Human Aurora B KD (60 ng), INCENP and Cdc7/ASK (25 ng) were incubated in kinase assays with [γ-32P] ATP. Increasing concentrations of a Cdc7 inhibitor (PHA-767491) were added, as shown. A long exposure of the autorad panel is shown in Supplementary Fig. S8.
