Abstract
BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, we demonstrate that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. We find that the BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, our results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Animals
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Cdc20 Proteins
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Survival
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Cells, Cultured
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Chromosomes / metabolism
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Cyclin B / genetics
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Cyclin B / metabolism*
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Fibroblasts / cytology
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Fibroblasts / physiology
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Humans
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Interphase
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Mice
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Mice, Knockout
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Spindle Apparatus / metabolism
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Tumor Suppressor Protein p53 / genetics
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Tumor Suppressor Protein p53 / metabolism
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Ubiquitin-Protein Ligase Complexes / metabolism*
Substances
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Bub1b protein, mouse
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Cdc20 Proteins
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Cdc20 protein, mouse
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Cell Cycle Proteins
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Cyclin B
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Recombinant Fusion Proteins
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Tumor Suppressor Protein p53
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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BUB1 protein, human
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Bub1 spindle checkpoint protein
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Protein Serine-Threonine Kinases