藉由受質 HURP 闡明致癌基因 Aurora-A 的受質特性
Elucidation the role of an oncogene Aurora-A through its
downstream substrate, HURP
中文摘要
Aurora 是一種 serine/threonine 激酶，在最近幾年的研究中發現與人類癌症發生及 細胞有絲分裂的過程有關。 在大腸直腸癌細胞中發現 Aurora-A 高量表現的情 形，將 Aurora-A 過量表現於細胞株可使 Rat-1 及 NIH 3T3 發生轉型，因此推測 Aurora-A 激酶為致癌基因。除此之外，Aurora-A 在細胞週期中的有絲分裂期表 現量達到最高，主要表現的區域位於紡錘體上。所以我們想要研究的關鍵問題是 Aurora-A 的下游受質究竟為何？這些受質之間是否存在專一的 motif，促使 Aurora-A 走向不同的訊息傳導路徑。在此研究中，我運用大量快速之 SPOTs 合 成技術及月生月太合成來找尋 Aurora-A 對受質磷酸化的 motif，實驗結果顯示 Aurora-A 與 PKA 對選擇專一受質具有類似的決定 motif，[K/R]-X-[S/T]或 [K/R]-[K/R]-X-[S/T]。另外，Aurora-A 具有一特殊的受質決定 motif [K/R]-[K/R]-X-[S/T]-[I/L/V]，此 motif 與酵母菌的 IPL1 的辨識 motif
[K/R]-X-[S/T]-[I/L/V]相似。一個由細胞週期所調節的 HURP 基因，與 Aurora-A 有相類似的特性，且在細胞外的實驗中顯示 HURP 為 Aurora-A 的受質。為了進 一步探討在細胞內 Aurora-A 與 HURP 的關聯，我利用 LC MS/MS 及定點突變定 義出可能的磷酸化位置。這是第一篇有系統地報導 Aurora-A 對受質磷酸化位置 的保留序列，並由此結果預測 Aurora-A 對受質 HURP 可能的磷酸化位置。 英文摘要
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.
