Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1.
The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.
Davenport
James
J
Department of Pathology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA.
Harris
Loleta D
LD
Goorha
Rakesh
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D064199:Cdc20 Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D008941:Spindle Apparatus / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
C584278:Bub1b protein, mouse; D064199:Cdc20 Proteins; C577491:Cdc20 protein, mouse; D018797:Cell Cycle Proteins; D064246:Mad2 Proteins; C577698:Mad2l1 protein, mouse; C472089:Mxd3 protein, mouse; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases