Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex.
The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.
Fang
Guowei
G
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA. gwfang@stanford.edu
D064173:Anaphase-Promoting Complex-Cyclosome; D000970:Antineoplastic Agents / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D064199:Cdc20 Proteins; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002842:Chromatids / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D015739:Nocodazole / Q000494:pharmacology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes
D000970:Antineoplastic Agents; C577495:CDC20 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D008025:Ligases; D015739:Nocodazole