PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme (GBM) is an aggressive, genetically diverse, and universally lethal malignancy of the brain. The aggressive characteristics and genetic diversity observed in GBM have been linked to a population of cells with stem-like characteristics known as glioblastoma stem cells (GSCs). In collaboration with the Rich lab, we have shown that EYA2 (eyes absent transcriptional coactivator and phosphatase 2) is highly expressed in GSCs compared to differentiated glioblastoma cells (DGCs) and neural stem cells (NSCs), where it is highly localized to centrosomes. Importantly, GSCs are uniquely reliant on EYA2’s tyrosine phosphatase (Tyr Ptase) activity for proliferation, as pharmacological inhibition of EYA2’s Tyr Ptase activity or overexpression of an EYA2 Tyr Ptase dead mutant causes cell cycle arrest, mono- and multipolar spindle abnormalities, and apoptosis when compared to DGCs and NSCs. Together, these data provide evidence that the Tyr Ptase activity of EYA2 elicits a novel function at centrosomes that is required for GSC proliferation. We have previously demonstrated that several cancers re-express EYA proteins to promote tumorigenic and metastatic phenotypes through multiple cytoplasmic and nuclear functions. The Tyr Ptase activity of EYA proteins is implicated in cell cycle progression in several cancers, but the molecular cause of these phenotypes is mostly unknown. Intriguingly, EYA proteins have not previously been shown to function at centrosomes. My data highlights that EYA2 is localized to centrosomes in GSCs and that inhibition of EYA2’s Tyr Ptase activity leads to centrosome fragmentation in mitotic GSCs, suggesting a novel, mitotic regulatory role for EYA2 in cancer. Furthermore, using EYA2 substrate-trapping experiments I have identified several centrosome- associated proteins and mitotic regulators as potential substrates of EYA2’s Tyr Ptase activity. Because the precise mechanism for EYA2’s Tyr Ptase activity at centrosomes and in mitotic spindle formation remains elusive, in this proposal I will test the hypothesis that EYA2’s tyrosine phosphatase activity is directly required at centrosomes for centrosome maturation and mitotic spindle formation, and that direct targets of the EYA2 Tyr Ptase activity can be identified that are critical for mitotic progression.