DISCOVERY OF AURORA-A KINASE PHYSIOLOGICAL SUBSTRATES IN PROSTATE CANCER CELLS

Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in multiple malignancies including prostate cancer, which is the most frequently diagnosed malignancy in men and the second leading cause of cancer death in the United States. Aurora-A is a key regulator of mitosis that localizes to the centrosome from the G2 phase of the cell cycle through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora-A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein Nuclear Mitotic Apparatus (NuMA) as a robust substrate of Aurora-A kinase. Using a small molecule Aurora-A inhibitor in conjunction with a Reverse In-gel Kinase Assay, we demonstrate that NuMA becomes hypo-phosphorylated in vivo upon Aurora-A inhibition. Using an alanine substitution strategy, we identified multiple Aurora-A phospho-acceptor sites in the C-terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these Aurora A phospho-acceptor sites significantly diminishes cell proliferation. In addition, alanine mutation at these sites significantly increases the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis-localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora-A phosphorylation sites in NuMA that play a role in cell cycle progression and apoptosis provides new insights into Aurora-A function