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Identification of an overlapping binding domain on Cdc20 for Mad2 and anaphase-promoting complex: model for spindle
1Department of Oncology, DNAX Research Institute, Palo Alto, California 94304-1104, USA.
Abstract:
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.
Insights
The anaphase-promoting complex (APC) regulates mitosis. Disrupting the Mad2-Cdc20 interaction prematurely activates APC, sensitizing cells to microtubule drugs and perturbing the mitotic checkpoint.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The anaphase-promoting complex (APC) is crucial for cell cycle progression, specifically regulating anaphase initiation and mitotic exit in mammalian cells.
- Cdc20 is a key activator of the APC, targeting substrates essential for the metaphase-to-anaphase transition.
- The spindle checkpoint, involving Mad2, inhibits APC-Cdc20 complex formation to prevent premature anaphase.
Purpose of the Study:
- To identify the specific regions of Cdc20 responsible for its interaction with Mad2 and the APC.
- To elucidate the structural basis for Mad2's modulation of the APC-Cdc20 complex.
- To investigate the functional consequences of disrupting the Mad2-Cdc20 interaction on mitotic progression and cell viability.
Main Methods:
- Deletion mutagenesis was employed to map interaction domains within Cdc20.
- Peptide mapping was used to pinpoint binding sites for Mad2 and the APC on Cdc20.
- Functional studies involved overexpressing a Cdc20 truncation mutant (1-153) and assessing its effects on cell morphology and drug sensitivity.
Main Results:
- Distinct but overlapping sequences in Cdc20 were identified for Mad2 and APC binding.
- A Cdc20 truncation mutant (1-153) was found to bind Mad2 constitutively but not the APC.
- Overexpression of this mutant led to multinucleated cells and increased sensitivity to microtubule-inhibiting drugs.
- Disruption of the Mad2-Cdc20 interaction resulted in premature APC activation and mitotic checkpoint perturbation.
Conclusions:
- The findings provide a structural explanation for Mad2's regulation of the APC-Cdc20 complex.
- Perturbing the Mad2-Cdc20 interaction compromises the mitotic checkpoint, leading to aberrant APC activation.
- This premature APC activation sensitizes cells to microtubule-targeting agents, highlighting the importance of this interaction for maintaining genomic stability.
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