BubR1 phosphorylates itself. (A) BubR1 kinase activity, as well as BubR1 autophosphorylation, is directly stimulated by CENP-E in vitro. Equal amounts of purified recombinant Xenopus BubR1 with (lanes 2 and 4) or without (lanes 1 and 3) purified recombinant Xenopus CENP-E were analyzed for BubR1 autokinase activity. (left) Autoradiography of SDS-PAGE gel of purified recombinant BubR1 autophosphorylated, as well as purified recombinant CENP-E phosphorylated, in the presence of γ-[³²P]ATP. (right) Coomassie blue staining of purified recombinant BubR1 and CENP-E proteins. (B) BubR1 protein structure showing the relative positions of KEN box, the N-terminal Mad3-like domain, the Bub3-binding domain, the kinase domain, and the autophosphorylation site identified by LC-MS/MS, which is conserved in frog (xBubR1), human (hBubR1), and mouse (mBubR1). The autophosphorylation site is shown in red in the DNA sequences. P, phosphorylated. (C–G) Immunofluorescence images acquired using the indicated antibodies against BubR1, the autophosphorylation site (pT608), and anticentromere antigen (ACA; a centromere/kinetochore marker) in nocodazole-treated human T98G cells with endogenous BubR1 (C), with endogenous BubR1 depleted (D), replaced with siRNA-resistant mCherry-tagged wild-type (WT) BubR1 (E), the nonphosphorylatable BubR1T608A mutant (F), or kinase-inactive (KD) BubR1 (G). Bar, 10 µm.