Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint.
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.
Braunstein
Ilana
I
Unit of Biochemistry, The Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Miniowitz
Shirly
S
Moshe
Yakir
Y
Hershko
Avram
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D064173:Anaphase-Promoting Complex-Cyclosome; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002457:Cell Extracts; C022571:adenosine 5'-O-(3-thiotriphosphate); D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D015739:Nocodazole