Checkpoint complexes in the cell cycle. HeLa cells were synchronized at the G1/S boundary by a double thymidine block and cells were collected at indicated time after being released from the arrest (lanes 1–6). Alternatively, HeLa cells were synchronized at prometaphase by a thymidine-nocodazole block. Cells were collected immediately after nocodazole treatment (lane 7), or released into fresh media for 1 and 2 h (lanes 8 and 9). (A) Levels of various mitotic regulators and checkpoint proteins were determined by Western blot analyses. (B) Cdc2 kinase was immunopurified from cell lysates and assayed using histone H1 as a substrate. (C–E) Lysates of synchronized HeLa cells were immunoprecipitated with anti-Cdc20 (C), anti-Mad2 (D), or anti-Cdc27 (E) antibody, and the immunoprecipitates were analyzed by Western blotting with anti-BubR1, Bub3, Mad2, Mad1, Cdc20, and APC2 antibodies. Arrowhead in D, a partial degradation product of Mad1.

BubR1 directly inhibits activation of APC by Cdc20, but not by Cdh1. (A) Recombinant BubR1 was expressed in Sf9 cells (lane 2) and purified to homogeneity (lane 3). Lane 1, Sf9 cell lysates without expressing BubR1. (B) BubR1 inhibits Cdc20. Interphase APC (iAPC) was incubated with purified recombinant Cdc20, Cdc20 plus BubR1, or Cdc20 plus Mad2 and the ligase activity of APC was measured using a radioactive N-terminal fragment of cyclin B as a substrate. The recombinant Mad2 protein exists in two different forms (oligomer and monomer) and only the oligomer is an active inhibitor of APC (Fang et al., 1998b). Thus, Mad2 oligomer was used in experiments described in this article. (C) BubR1 does not inhibit Cdh1. Interphase APC (lane 1) was incubated with purified recombinant Cdc20 (lane 2), with Cdc20 plus BubR1 (lane 3), with Cdh1 (lane 4), or with Cdh1 plus BubR1 (lane 5). The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. (D) BubR1 does not inhibit mitotic APC. Mitotic APC was incubated with a buffer (lanes 1 and 4), BubR1 (lanes 2 and 5), or with Mad2 (lanes 3 and 6) either in the presence (lanes 4–6) or absence (lanes 1–3) of Cdc20, and the ubiquitin ligase activity of APC was measured using the N-terminal fragment of cyclin B as a substrate. (E) BubR1 does not inhibit preactivated Cdc20-APC. Interphase APC beads (lane 1) were first incubated with recombinant Cdc20 (lanes 2 and 3). BubR1 was then added and incubated with the mixture of Cdc20 and APC (lane 3). The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. (F) BubR1 and Mad2 do not directly inhibit APC. Recombinant BubR1 (lane 2) or Mad2 (lane 3) was incubated with iAPC in the presence of ATP. BubR1 and Mad2 were then removed and APC beads incubated with recombinant Cdc20. The ubiquitin ligase activity of APC was measured.

Phosphorylation of Cdc20 is not required for inhibition by BubR1. (A) Cell cycle-dependent phosphorylation of Cdc20 by the BubR1 immunocomplex. Synchronous HeLa cells (lanes 1–9) were prepared as described in Figure 1. BubR1 was immunopurified from cell lysates and assayed for phosphorylation of Cdc20 in the presence of radioactive γ-ATP. Lane 10, Cdc20 alone in the absence of BubR1. (B–D) Effect of phosphorylation on Cdc20 activity. BubR1 was immunopurified from double thymidine-arrested cells (G1/S, lane 2) or from thymidine-nocodazole–arrested cells (M, lane 3). BubR1 beads were incubated with recombinant Cdc20 in a kinase buffer containing radioactive γ-ATP, and phosphorylated Cdc20 was analyzed by SDS-PAGE (B). In parallel reactions, BubR1 beads were incubated with recombinant Cdc20 and nonradioactive ATP (C and D). After removal of BubR1 beads, one-tenth of Cdc20 was analyzed by Western blotting to determine its mobility (C) and the remaining Cdc20 was incubated with immunopurified iAPC. The ubiquitin ligase activity of APC was then assayed for 10 min and 150 min. Lane 1, iAPC alone. (E) Interphase APC was incubated with recombinant BubR1 (lane 3) or Mad2 (lane 4) in the presence (lanes 2–4) or absence (lane 1) of Cdc20. The ubiquitin ligase activity of APC was then analyzed in the presence of AMP-PNP, not ATP.

BubR1 and Mad2 act synergistically to inhibit activation of APC by Cdc20. (A and B) Dose response of BubR1 (A) and Mad2 (B) in inhibition of Cdc20. Decreasing amounts of recombinant BubR1 and Mad2 were incubated with constant amounts of Cdc20 and iAPC. The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. Lane 1 in A: interphase APC without Cdc20. Lane 2 in A: Cdc20-APC in the absence of BubR1. (C-E) Synergism between BubR1 and Mad2 in inhibition of Cdc20. Various amounts of recombinant BubR1 and Mad2 were incubated with constant amounts of Cdc20 and iAPC. The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. Ubiquitination reactions were done in the presence of ATP (C and D) or in the presence of AMP-PNP (E). Data in A–D were generated in a single experiment and gels were exposed to the same extent. Therefore, APC activity in different panels is directly comparable.

Mad2B and Bub3 do not have synergistic effect with BubR1 in inhibiting Cdc20. (A) Dose response of Mad2B in inhibition of Cdc20. Decreasing amounts of recombinant Mad2B were incubated with constant amounts of Cdc20 and iAPC. The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. (B) Various amounts of recombinant Mad2B were incubated with constant amounts of BubR1, Cdc20, and iAPC. The ability of treated APC to ubiquitinate the radioactive N-terminal fragment of cyclin B was assayed. (C) Interphase APC (lane 1) was incubated with recombinant Cdc20 (lane 2), Cdc20 plus BubR1 (lane 3), Cdc20 plus Bub3 (lane 4), or Cdc20 plus BubR1 and Bub3 (lane 5), and the ubiquitin ligase activity of treated APC was assayed.

Protein–protein interactions among BubR1, Cdc20, and Mad2. (A) Purified recombinant Cdc20 and BubR1 directly interact with each other. Recombinant Myc-Cdc20 was incubated with BubR1 and then immunoprecipitated with an anti-Myc antibody (lane 2) or with control IgG (lane 3). As a control, BubR1 was immunoprecipitated with the anti-Myc antibody in the absence of Myc-Cdc20 (lane 1). (B) Mad2 enhances interactions between BubR1 and Cdc20. Myc-BubR1 was incubated with increasing amounts of recombinant Mad2 in the presence (lanes 2, 4, 6, 8, 10, and 12) or absence (lanes 3, 5, 7, 9, 11, and 13) of labeled HA-Cdc20. Protein complexes were immunoprecipitated by an anti-HA antibody and associated Myc-BubR1 protein analyzed by SDS-PAGE. Lane 1, one-tenth of input Myc-BubR1 in the binding reaction. (C) The amount of bound Myc-BubR1 from A was quantitated, normalized with the amount of HA-Cdc20 immunoprecipitated, and plotted against Mad2 concentration (squares). The unit for the amount of bound Myc-BubR1 is arbitrary. Similarly, the interaction between Myc-BubR1 and HA-Cdc20 was analyzed in the presence of Mad2ΔC and the amount of bound BubR1 was plotted against Mad2ΔC concentration (circles). (D) Interactions among BubR1, Cdc20, and Mad2. Myc-Cdc20 (lanes 1–3), Myc-BubR1 (lanes 4–6), or Myc-Cdc20 plus Myc-BubR1 (lanes 7–9) were translated in vitro. These proteins were incubated with 4 μM recombinant Mad2 and the Mad2 complexes were immunoprecipitated by an anti-Mad2 antibody (lanes M) or by control IgG (lanes G). The amount of bound Myc-Cdc20 and Myc-BubR1 was analyzed by SDS-PAGE. Lanes 3, 6, and 9, immunoprecipitation of Myc-Cdc20 and Myc-BubR1 by the anti-Mad2 antibody in the absence of recombinant Mad2. Lanes 10 and 11, one-tenth of the input Myc-Cdc20 and Myc-BubR1 used in the binding reactions. (E) BubR1 enhances interactions between Mad2 and Cdc20. HA-Cdc20 and Myc-Mad2 were incubated with increasing amounts of recombinant BubR1. Myc-Mad2 was immunoprecipitated by an anti-Myc antibody (lanes 2, 4, 6, 8, 10, 12, 14, and 16) or by control IgG (lanes 3, 5, 7, 9, 11, 13, 15, and 17) and the amount of associated HA-Cdc20 protein was analyzed by SDS-PAGE. Lane 1, one-tenth of the input HA-Cdc20 in the binding reactions.

Checkpoint complexes in nocodazole-arrested HeLa cells. (A) Mitotic checkpoint was activated in HeLa cells by a thymidine-nocodazole arrest. Cell lysates were fractionated through a Resource Q column and fractionation profiles of BubR1, Cdc20, APC2, and Mad2 were determined by immunoblotting. (B) Fractions from peak A were pooled, concentrated, and further fractionated by a Superose 6 column. Fractionation profiles of BubR1, Bub3, Cdc20, Mad2, and Mad1 were determined by immunoblotting. Arrowhead, a partial degradation product of Mad1. (C–E) Fractions 25, 29, and 37 from the Superose 6 column were immunoprecipitated with anti-Mad2 (C), anti-Cdc20 (D), and anti-BubR1 (E) antibodies, followed by Western blot analyses with antibodies against various checkpoint proteins.