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MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity
1Cell Biology Program, Memorial-Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Abstract:
Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.
Insights
The mitotic checkpoint protein MAD2 binds to the anaphase-promoting complex, inhibiting cyclin B destruction. This discovery reveals how MAD2 halts cell division when chromosomes are improperly aligned, ensuring accurate genome segregation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Cell cycle progression relies on checkpoint mechanisms for accurate genome duplication and segregation.
- The mitotic checkpoint prevents cell cycle progression upon detecting errors in spindle assembly or kinetochore attachment.
- This checkpoint arrests cells before the metaphase-anaphase transition, maintaining chromosome integrity.
Purpose of the Study:
- To elucidate the molecular mechanisms by which the mitotic checkpoint controls cell cycle progression.
- To investigate the role of MAD2 in regulating the metaphase-anaphase transition.
- To identify the interaction partners of MAD2 during mitotic arrest.
Main Methods:
- Biochemical assays using Xenopus laevis egg extracts.
- Purification of MAD2 protein.
- Analysis of cyclin B ubiquitination and degradation.
- Observation of cell cycle arrest at metaphase.
Main Results:
- MAD2 (an essential mitotic checkpoint component) associates with the anaphase-promoting complex (APC/C), a cyclin B-ubiquitin ligase.
- Purified MAD2 induces metaphase arrest in Xenopus egg extracts.
- MAD2 inhibits cyclin B proteolysis by blocking its ubiquitination.
- MAD2 directly links the mitotic checkpoint to the cyclin B destruction machinery.
Conclusions:
- MAD2 acts as a direct inhibitor of the anaphase-promoting complex.
- This inhibition by MAD2 is crucial for arresting the cell cycle at metaphase.
- The findings clarify how the mitotic checkpoint prevents premature anaphase onset and ensures accurate chromosome segregation.