title: Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding.
abstract: Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.
authors.affiliation: Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Kulukian
authors.forename: Joo Seok
authors.initials: JS
authors.lastname: Han
authors.forename: Don W
authors.initials: DW
authors.lastname: Cleveland
mesh_terms: D000705:Anaphase / Q000502:physiology*; D064173:Anaphase-Promoting Complex-Cyclosome; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C489444:BUB3 protein, human; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases