藉由受質 HURP 闡明致癌基因 A urora-A 的受質特性

藉由受質 HURP 闡明致癌基因 A urora-A 的受質特性

 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 m otif ， [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 M S/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 Auror a-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。

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

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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 ide ntified from screening for overexpressed kinase in colon carcinoma. Ectopic expression of Aur ora-A transforms Rat-1 and NIH3T3 cells, suggesting that the kinase possesses oncogenic pot ential. In addition, Aurora-A is overexpressed in mitosis and exhibits unique subcellular locali zation, namely mitotic spindle in mitosis. The key questions are what are the downstream subs trates and whether these substrates exhibit unique recognition motifs for Aurora-A to propagat e 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 s uggests 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 sp

ecificity 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 characteri

stics 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-dir

ected 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 an

d reveals the possible phosphorylation sites of HURP by Aurora-A kinase.