Wong OK , Fang G .

GM062852 NIGMS NIH HHS , R01 GM062852 NIGMS NIH HHS , Wellcome Trust

	Figure 1. Cdk1- and Plx1-mediated phosphorylation of BubR1 generated the 3F3/2 epitope. (A) BubR1 was immunoprecipitated (IP) from either checkpoint extracts (lanes 1 and 2) or CSF extracts (lanes 3 and 4) and treated with (lanes 1 and 4) or without (lanes 2 and 3) λ-phosphatase. Control immunoprecipitation was performed in CSF extracts using nonspecific rabbit IgG (lane 5). Samples were assayed by SDS-PAGE, and identical regions of the duplicated blots were analyzed by anti-3F3/2 and -BubR1 antibodies. (B) Immunoprecipitates of control or BubR1 antibodies from CSF extracts were analyzed by Western blotting with anti-Plx1 and -BubR1 antibodies. (C and D) Chromosomes purified from checkpoint extracts were dephosphorylated by λ-phosphatase, treated with NEM, and subsequently rephosphorylated (ReP) by incubation with ATP alone or ATP plus Plx1 and/or Cdk1/cyclin B. Phosphorylated chromosomes were stained for the indicated antigens (C), and mean fluorescence intensities of kinetochore 3F3/2 signals (n = 20 kinetochores) were quantified and normalized to the corresponding value derived from samples rephosphorylated by both Plx1 and Cdk1/cyclin B (D). Error bars represent SEM. Bar, 5 μm. (E) Recombinant GST-BubR1 and GST-BubR1-T605A were first incubated at 25°C for 75 min, with or without Cdk1/cyclin B, in the presence of 0.2 mM of unlabeled ATP. Samples were then split equally and incubated for 40 min in the presence of purvalanol A with γ-[32P]ATP alone (middle) or γ-[32P]ATP plus Plx1 (top). The amounts of recombinant GST-BubR1 and GST-BubR1-T605A from duplicated samples were shown by Coomassie blue (CB) staining. The amount of 32P incorporated in the top panel was quantified and plotted. Compared with BubR1, more BubR1-T605A was used in the reaction, which explains more efficient phosphorylation of BubR1-T605A by Plx1 in the absence of Cdk1. (F and G) 1 μg each of recombinant GST-BubR1, GST-BubR1-T605A, and GST-BubR1 kinase-dead mutant (KD; K788R) were incubated with the indicated kinases in the presence of 0.2 mM ATP for 2 h, and then subjected to SDS-PAGE followed by Western blotting with the 3F3/2 antibody. (H) 3 μg each of recombinant GST-BubR1, GST-BubR1-T605A, GST-BubR1-T605E, and GST control were incubated with 140 μl CSF extracts for 20 min, immunoprecipitated with an anti-GST antibody, and then subjected to Western blotting for the associated Plx1. (I and J) Chromosomes purified from checkpoint extracts that had undergone immunodepletion (ID) and addback (AB) of equal amounts of the indicated proteins were dephosphorylated (De-P) by λ-phosphatase, treated with NEM, and subsequently rephosphorylated by Plx1 and Cdk1/cyclin B. Phosphorylated chromosomes were stained for indicated antigens (I), and mean fluorescence intensities of kinetochore 3F3/2 (red) and BubR1 (green; n = 20 kinetochores) were quantified and normalized to the corresponding value derived from mock-depleted extracts (J). Error bars represent SEM. Bar, 5 μm.
	Figure 2. Thr 605 in BubR1 is required for spindle checkpoint arrest. (A) CSF extracts were mock depleted (lane 5) or depleted (lanes 1–4) of BubR1. Recombinant GST-tagged BubR1 (lane 2), BubR1-T605A (lane 3), or BubR1-T605E (lane 4) was added to the depleted extracts. Different amounts of extracts were loaded to determine the depletion efficiency. (B and C) CSF extracts that had undergone immunodepletion and addback of the indicated proteins were incubated with sperm chromosomes and nocodazole to activate the spindle checkpoint, followed by incubation with (top) or without (bottom) calcium. At the indicated times, an aliquot of extracts was assayed for the Cdk1 kinase activity using histone H1 as a substrate. The kinase activity was quantified and plotted (C) upon normalization to the value at time 0 for the corresponding samples. C shows mock-depleted extracts (star), BubR1-depleted extracts (square), BubR1-depleted extracts with addback of GST-BubR1 (filled circle), GST-BubR1-T605A (open circle), and GST-BubR1-T605E (triangle). (D and E) Inhibition of APC-Cdc20–mediated ubiquitination by equal amounts of recombinant GST-BubR1 and GST-BubR1-T605A was assayed using in vitro translated 35S-securin substrate, as described previously (Fang, 2002). The amount of securin remaining was quantified and plotted (E) upon normalization to the value at time 0 for the corresponding samples. E shows interphase APC (iAPC; square), iAPC + Cdc20 (filled circle), iAPC + Cdc20 + BubR1 (triangle), and iAPC + Cdc20 + BubR1-T605A (open circle).
	Figure 3. Thr 605 in BubR1 is required for the recruitment of Plx1 and Mad2 to kinetochores. Chromosomes were purified onto coverslips from checkpoint extracts that had undergone immunodepletion and addback of the indicated proteins, as prepared in Fig. 2 A. Purified chromosomes were stained in green for Mad2 (A), Plx1 (C), and Mps1 (E). DNA was stained in blue. Mean fluorescence intensities of Mad2 (B), Plx1 (D), and Mps1 (F) were quantified from 20 kinetochores in different fields and plotted upon normalization to the corresponding values derived from mock-depleted extracts. Error bars represent SEM. Bars, 5 μm.
	Figure 4. Activation of the BubR1 kinase by Plx1 and Cdk1 is dependent on Thr 605. (A) BubR1 autophosphorylation was assayed as diagramed. Recombinant GST-BubR1 was prephosphorylated (Pre-P) in 0.2 mM of unlabeled ATP in the presence (sample 2) or absence (sample 1) of Plx1 and Cdk1/cyclin B. Plx1 was then removed by anti-Plx1 antibody beads, and prephosphorylated BubR1 was assayed for its autophosphorylation activity in the presence of γ-[32P]ATP and purvalanol A. In a parallel control (sample 3), recombinant GST, not BubR1, was used in the prephosphorylation reaction, and thereafter an aliquot of unphosphorylated GST-BubR1 protein was added before the autophosphorylation reaction. (B) 550 ng each of wild-type BubR1 and kinase-dead mutant was assayed for autophosphorylation after being prephosphorylated by Cdk1 and Plx1 as described in A. (C and D) 550 ng each of recombinant GST-BubR1 (lanes 1–4), GST-BubR1-T605A (lanes 8–11), and GST (lanes 5–7 and 12–14) was prephosphorylated by either Cdk1/cyclin B (lanes 2, 5, 9, and 12) or Plx1 (lanes 3, 6, 10, and 13), or by both kinases (lanes 4, 7, 11, and 14). The autophosphorylation assay was then performed as described in A. In control GST samples (lanes 5–7 and 12–14), unphosphorylated BubR1 (lanes 5–7) or BubR1-T605A (lanes 12–14) were added after the prephosphorylation step but before the autophosphorylation reaction. The extent of autophosphorylation was quantified by measuring the amount of 32P incorporated in BubR1 (D).

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