Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

Abstract

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925-936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5'-(gamma-thio)triphosphate (ATP[gammaS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration.

Fig. 1.

Existence of labile inhibitor(s) of APC/C in extracts from nocodazole-arrested cells. (A) Lag kinetics of the degradation of [³⁵S]securin in extract from nocodazole-arrested cells and influence of preincubation of extract. The degradation of ³⁵S-labeled securin was determined as described in Methods, in the presence of either untreated extract (lanes 1–4) or of extract that had been preincubated (23°C, 3 h) with an ATP regeneration mixture (lanes 5–8). (B) Inhibition of the degradation of [³⁵S]securin in preincubated extract by untreated extract. The degradation of [³⁵S]securin was determined in the presence of untreated extract (filled squares), preincubated extract (filled triangles), or a 1:1 mixture of untreated and preincubated extracts (open triangles).

Fig. 2.

Protein phosphorylation is required for maintenance of the checkpoint-arrested state of APC/C. (A) Influence of staurosporine on the kinetics of the degradation of [³⁵S]securin in extracts from nocodazole-arrested cells. The kinetics of [³⁵S]securin degradation was determined in the presence (filled circles) or absence (open circles) of 10 μM staurosporine. (B) Preincubation of extracts with the ATP analogue ATP[γS], but not with AMP-PNP, stabilized the checkpoint-arrested state. Extracts from nocodazole-arrested cells were either untreated or preincubated (23°C, 3 h) with 2 mM ATP, ATP[γS], or AMP-PNP, as indicated, before the supplementation of [³⁵S]securin and “degradation mixture” (see Methods).

Fig. 3.

Inhibitory factor from checkpoint-arrested extract is associated with APC/C and can be dissociated with increased salt concentration. (A) Ubiquitinylation of [³⁵S]securin by APC/C immunoprecipitated from checkpoint-arrested and activated extracts. “Arrested” and “activated” extracts were prepared, and adsorption of APC/C to anti-Cdc27 beads was carried out as described in Methods. The ligation of [³⁵S]securin to ubiquitin was carried out as described in Methods, with samples of 1-μl anti-Cdc27 beads, for the time periods indicated. Where indicated, 2 ng of recombinant Cdc20 was added. (B) Ubiquitinylation activity of APC/C immunoprecipitated from checkpoint-arrested extracts is stimulated by high-salt wash. Adsorption to anti-Cdc27 beads, followed by washes with Buffer A was carried out as described in Methods. Subsequently, beads were washed two more times with either Buffer A (open bars), or Buffer A that contained 0.3 M KCl (filled bars). Ubiquitinylation of [³⁵S]securin was estimated in samples of 1 μl of anti-Cdc27 beads incubated for 20 min at 20°C in the presence of 2 ng of Cdc20. (C) Salt eluate of APC/C immunoprecipitate from checkpoint-arrested extracts inhibits cyclin–ubiquitin ligation by purified APC/C^Cdc20. Salt eluates of anti-Cdc27 immunoprecipitates of checkpoint-arrested and activated extracts were prepared, and the ubiquitinylation of [¹²⁵I]-cyclin was assayed as described in Methods. Numbers on the right indicate the position of molecular mass marker proteins (in kilodaltons).

Fig. 4.

MCC accounts for a part of APC/C-associated inhibitors. (A) Presence of BubR1, Cdc20, and Mad2 in salt eluates of APC/C immunoprecipitates from checkpoint-arrested and activated extracts. Samples of 5 μl of these preparations (see Methods) were separated on a 10% polyacrylamide-SDS gel and blotted with the indicated antibodies. (B) Immunodepletion by anti-Cdc20 of salt eluate of APC/C immunoprecipitate from checkpoint-arrested extract. Samples of 30 μl of salt eluates were treated with 10 μl of Affi-prep Protein A beads to which 10 μg of affinity-purified polyclonal anti-Cdc20 antibody (provided by Tim Yen) or no antibody (“Sham”) had been previously adsorbed. After rotation at 4°C for 2 h, beads were removed by centrifugation. Samples of 5 μl of supernatants were subjected to immunoblotting as described above. (C) Immunodepletion by anti-Cdc20 removes a part of inhibitory activity. The indicated amounts of the preparations described in (B) were added to cyclin ubiquitinylation assay in the presence of purified APC/C and Cdc20. (D) MCC disassembled in extract incubated with ATP but not with ATP[γS]. Samples of 5 μg of polyclonal anti-Cdc20 antibody (sc-8358; Santa Cruz Biotechnologies) were adsorbed to 10 μl of Affi-prep Protein A beads, and then beads were mixed with 40 μl of either untreated extract from nocodazole-arrested cells (lane 1) or extracts that had been incubated with either ATP (lane 2) or ATP[γS], as described in Methods. After rotation at 4°C for 2 h, beads were washed three times with Buffer A and then were suspended in 40 μl of Buffer A that contained 10 mg/ml BSA. Samples of 5 μl of bead suspension were boiled with SDS electrophoresis sample buffer, separated on 10% polyacrylamide-SDS gel, and blotted with the indicated antibodies.