Elucidation the role of an oncogene Aurora-A through its downstream substrate, HURP

• Main Authors: Huang Yin-Jou, 黃尹柔
• Other Authors: Hung Li, Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/64641989475226406662

碩士 === 臺北醫學大學 === 細胞及分子生物研究所 === 91 === Aurora, an emerging family of serine/threonine kinases, has drawn intensive attention recently for the correlation with human cancer development and mitotic progression. Aurora-A was identified from screening for overexpressed kinase in colon carcinoma. Ectopic expression of Aurora-A transforms Rat-1 and NIH3T3 cells, suggesting that the kinase possesses oncogenic potential. In addition, Aurora-A is overexpressed in mitosis and exhibits unique subcellular localization, namely mitotic spindle in mitosis. The key questions are what are the downstream substrates and whether these substrates exhibit unique recognition motifs for Aurora-A to propagate diverse signaling pathways. In this study, I have investigated the phosphorylation site motifs for Aurora-A by high throughput SPOTs synthesis followed by peptide synthesis. The result suggests that Aurora-A may exhibit very similar substrate specificity determinant, [K/R]-X-[S/T] or [K/R]-[K/R]-X-[S/T], to PKA. In addition, Aurora-A also exhibits a distinct substrate specificity determinant, [K/R]-[K/R]-X-[S/T]-[I/L/V], which is similar to yeast IPL1 [K/R]-X-[S/T]-[I/L/V]. Several properties of a novel cell cycle regulator, HURP, exhibit similar characteristics to Aurora-A and HURP serves as a substrate for Aurora-A in vitro. To further provide the in vivo correlation between Aurora-A and HURP, I have used LC MS/MS followed by site-directed mutagenesis to identify several potential phosphorylation sites. This is the first report of systematically search for the phosphorylation site consensus sequences of Aurora-A kinase and reveals the possible phosphorylation sites of HURP by Aurora-A kinase.