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藉由受質 HURP 闡明致癌基因 Aurora-A 的受質特性

藉由受質 HURP 闡明致癌基因 Aurora-A 的受質特性.

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藉由受質 HURP 闡明致癌基因 Aurora-A 的受質特性

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  1. 藉由受質HURP闡明致癌基因Aurora-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。

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

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