PROJECT SUMMARY Cell division is a complex process that is tightly regulated by the activation and inactivation of a series of proteins that ensure the proper segregation of genetic material into two daughter cells. Misregulation of key mitotic events, including chromosome alignment and segregation, can lead to genetic mutation that promotes abnormal cell proliferation and tumorigenesis. To better understand cell division and its misregulation in human disease, we performed a genetic RNAi screen for novel mitotic regulators and identified the phosphatase DUSP12. While DUSP12 has been shown to possess pro-survival attributes, it is unclear if this is related to its potential role in cell division. Preliminary proteomic analyses of DUSP12 using affinity- and proximity-based approaches coupled to mass spectrometry identified ZPR9 as a potential interactor of DUSP12 which has been validated by in-cell and in-vitro IP assays. Previous studies have shown that ZPR9 may play a role in apoptosis, but it is unclear if and how ZPR9 participates in cell division and, more so, how it promotes cell death. The central hypothesis of this project is that DUSP12 is a novel mitotic regulator and it influences cell fate by de-phosphorylating its target substrates, including ZPR9, which plays a critical role in the processes of cell death and proliferation. Aim 1 will characterize how DUSP12 modulation influences mitotic progression. Using cancer cell lines as a model system and leveraging advanced imaging techniques, we will explore the temporal and spatial activity of DUSP12 and its impact on key mitotic events and machinery. Aim 2 will evaluate the molecular mechanisms through which DUSP12 influences cell death and survival and if and how this applies to its role in cell division. These studies will bring new insights to our understanding of the importance of protein phosphatases for cell division, advancing our understanding of the full complement of proteins required for mitotic regulation and their relevance to human health and disease. Collectively, this work will provide insights into the mechanism and regulatory role of the phosphatase DUSP12 during cell division as well as form the basis for designing novel therapeutic approaches to address diseases associated with abnormal proliferation.