Abstract 1619: Discovering a new physiological substrate of Aurora-A kinase

Abstract 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 many cancers including prostate cancer, which is the most frequently diagnosed malignancy in men and second leading cause of cancer death in the United States. Aurora-A is one of the key regulators of mitosis that localizes to the centrosome from G2 phase through mitotic exit, where it regulates mitotic spindle formation and centrosome separation. Although Aurora-A is known to be an oncogene, few Aurora-A substrates have been identified. It is essential to elucidate Aurora-A substrates to develop biomarkers for Aurora-A inhibitors, and to identify new targets downstream of Aurora-A for cancer therapy. To identify new Aurora-A substrates, an Aurora-A Reverse In-gel Kinase Assay was developed. The basis of this method is co-polymerization of a denatured protein kinase throughout a polyacrylamide gel followed by protein refolding. Subsequently, an in-gel kinase reaction in the presence of γ-32P-ATP allows phosphorylation of potential kinase substrates that can be located by autoradiography. Phosphorylated proteins can be extracted from the gel and identified by liquid chromatography mass spectrometry. Using this method, we were able to identify Nuclear Mitotic Apparatus (NuMA) as an Aurora-A kinase substrate. Preliminary in vitro kinase experiments verify phosphorylation of recombinant NuMA C-terminus by Aurora-A. The identification of Aurora-A phosphorylation site/s will permit a better understanding of Aurora-A function in normal and cancer cells. The location of Aurora-A phosphorylation of NuMA and consequences of their mutation will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2011-1619