Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.
Sudakin
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V
Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Chan
G K
GK
Yen
T J
TJ
D064173:Anaphase-Promoting Complex-Cyclosome; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D005656:Fungal Proteins / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D018816:Genes, cdc / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000378:metabolism; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D008938:Mitosis / Q000502:physiology; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
C489444:BUB3 protein, human; C577495:CDC20 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C577695:MAD2 protein, S cerevisiae; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D008025:Ligases