Functional Analysis of Mitotic Checkpoint Gene Bub1b

St. Jude Children'S Research Hospital, Memphis TN

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Mitotic checkpoint proteins ensure accurate chromosome segregation into daughter cells by detecting errors in chromosome alignment and by delaying mitosis until the errors are corrected. Loss-of-function mutations in mitotic checkpoint genes cause aneuploidy, a hallmark of tumor cells and a key component of cancer pathogenesis. For example, haplo-insufficiency of mitotic checkpoint gene Mad2 causes chromosome missegregation leading to aneuploidy in mammalian cells and the development of lung tumors in mice. In addition, mutations in the human mitotic checkpoint gene BUB1B have been found in 10% of aneuploid colon cancers, suggesting that Bub1b may be involved in pathogenesis of some cancers. The murine homologue (Bub1b) of the yeast mitotic checkpoint protein Bub1 was discovered by the principal investigator and shown to be expressed in a cell cycle-specific manner. Because Bub1b is a mitotic checkpoint protein, it is possible that a loss of function of Bub1b may lead to aneuploidy. Before this possibility can be fully addressed, two objectives must be achieved: 1. Identification of Bub1b's functions during mitosis and cytokinesis; 2. Identification of Bub1b's functional domains. This application proposes studies to accomplish these objectives. The basis of cell cycle specific expression of Bub1b protein, especially aspects involving ubiquitination and phosphorylation, will be investigated (Specific aims 1 a and b). An engineering-based method will identify proteins that are phosphorylated by Bub1b and therefore likely to be in a Bub1b-activated pathway that institutes the mitotic checkpoint (Specific Aim 1c). The Principal Investigator has obtained evidence suggesting that Bub1b is required for proper chromosome movement during prophase and for cytokinesis. To identify Bub1b's domains responsible for these and other functions, mutations will be introduced into Bub1b, and each mutant's functions will be evaluated (Specific Aim 2a). Phase-contrast microscopy of mutant-expressing cells will identify the phases of mitosis and cytokinesis in which Bub1b is required (Specific Aim 2b). These studies are expected to establish the functions of Bub1b during mitosis and cytokinesis. [unreadable] [unreadable]