Accurate sister chromatid segregation during anaphase is pivotal for faithful transmission of genetic information during each cell division. Mistakes in this process result in missegregation of chromosomes and lead to aneuploidy which is often found in human cancers. The onset of anaphase is prohibited by the spindle assembly checkpoint in the presence of defects in the mitotic spindle or in chromosome attachment to the spindle, thus preventing premature anaphase from occurring. The long-term objective of this proposal is to understand at molecular and biochemical levels how the spindle assembly checkpoint regulates the metaphase to anaphase transition. The question will be approached biochemically in frog egg extracts using the key checkpoint component XMAD2 and its interacting protein p78 as the probes.
The specific aims are: (1) Characterization of p78 and its interaction with XMAD2. Immunodepletion of specific proteins or addition of modified forms of proteins will be performed in the egg extracts to determine the function of p78 in the spindle assembly checkpoint, the role of the p78-XMAD2 interaction, and the order of XMAD2 and p78 in the checkpoint signaling pathway. (2) Biochemical isolation of checkpoint components upstream of p78. The regulation of p78 phosphorylation during the cell cycle will be characterized. After the phosphorylation sites are determined, mutagenesis studies will be performed to determine their roles in the checkpoint. Phosphorylation of p78 will be used as a tool to purify the upstream kinase for p78 that may regulate the activity of the p78-XMAD2 complex. (3) Isolation of additional XMAD2-binding proteins. Affinity chromatography and the yeast two-hybrid assay will be used to isolate other XMAD2-binding proteins that are likely checkpoint molecules and may contribute to the constitutive checkpoint activity of excess XMAD2 and the dominant negative activity of a truncated XMAD2. In addition to the biochemical studies, yeast genetics will be used to analyze Mad2 in detail and to identify other spindle assembly checkpoint components by suppressor screens. These biochemical and genetic studies will provide a foundation for understanding the mechanisms of the spindle assembly checkpoint. The knowledge will then be applied to the question of how the deregulation of the checkpoint function may contribute to cancer progression.
