Principal Investigator/Program Director (Last, first, middle): Liu, Xuedong Faithful segregation of chromosomes during each cell division is normally ensured by the mitotic spindle checkpoint, which delays the onset of anaphase until every chromosome has successfully attached to the spindles. Defects in the mitotic checkpoint decrease genome stability and promote aneuploidy. Homozygous deletion of tumor suppressor Smad4 is frequently observed in aneuploid but not diploid colon cancer cells. Why Smad4 is so frequently deleted in tumor cells exhibiting chromosome instability is not well understood. The long term goal for this application is to understand the molecular mechanism(s) underlying the prevalence of chromosome instability exhibited in Smad4-deficient cancer cells. In searching for Smad4 partners in colon cancer cells, we discovered a novel function for Smad4 in mitosis. Our preliminary data suggest that Smad4 enhances the mitotic checkpoint response by targeting TTK/Mps1, a protein kinase required for normal mitotic progression and for triggering the mitotic checkpoint response. The overarching hypothesis of the current proposal is that the mitotic function of Smad4 is to prevent chromosome mis-segregation by allosterically modulating Mps1 kinase activation and phosphorylation. In doing so, Smad4 promotes kinetochore localization of Mps1 and robust mitotic checkpoint signaling. This project aims to test this hypothesis by pursuing the following specific aims: 1) Determine the role of phosphorylation in regulating Mps1 activation and whether phosphorylation of Mps1 is regulated by Smad4 association. 2) Determine whether Smad4 and Mps1 phosphorylation regulate kinetochore localization of Mps1 upon mitotic checkpoint activation. 3) Define the mechanisms for kinetochore localization of Mps1 upon mitotic checkpoint activation and identify Mps1 substrates necessary for its function in checkpoint signaling. Completion of these studies will provide important insights into novel mechanisms of non-canonical Smad signaling to control genome stability. We discovered the novel link between Smad4 and Mps1 through an unbiased proteomic screen and are well positioned to perform the proposed studies. It is expected that the proposed studies will be of high impact as knowledge gained will likely serve as a paradigm for this essential cellular process involving the targeting of checkpoint proteins to kinetochores. Elucidation of the detailed molecular mechanisms for how Smad4 regulates Mps1 in checkpoint signaling will likely yield insights critical to the development of new anticancer drugs to slow down tumor progression. Project Description Page 6