Identification of an overlapping binding domain on Cdc20 for Mad2 and anaphase-promoting complex: model for spindle checkpoint regulation.
Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.
Zhang
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Department of Oncology, DNAX Research Institute, Palo Alto, California 94304-1104, USA.
Lees
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D000595:Amino Acid Sequence; D000705:Anaphase; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D064246:Mad2 Proteins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
C577495:CDC20 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D015739:Nocodazole