title: Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint.
abstract: The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31(comet) (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31(comet) protein antagonizes the ability of Mad2 to inhibit APC/C(Cdc20) in vitro and in Xenopus egg extracts. Interestingly, p31(comet) binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31(comet) to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31(comet) forms a transient complex with APC/C(Cdc20)-bound Mad2. Purified p31(comet) enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31(comet) counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.
authors.affiliation: Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
authors.forename: Guohong
authors.initials: G
authors.lastname: Xia
authors.forename: Xuelian
authors.initials: X
authors.lastname: Luo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Habu
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Hongtao
authors.initials: H
authors.lastname: Yu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D064199:Cdc20 Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D008941:Spindle Apparatus / Q000378:metabolism*; D013379:Substrate Specificity; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; C470309:MAD2L1BP protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome