细胞有丝分裂调节激酶Aurora-A的自身活性调节与作用模式的研究

【作者】 张悦；

【导师】 赵寿元；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 Aurora是一类独特的丝/苏氨酸激酶,主要参与细胞的有丝分裂和减数分裂的调节。人类Aurora激酶家族的A、B、C三个成员在多种恶性肿瘤均显著上调表达,而且转基因实验证明它们对细胞具有转化作用,故它们被认为与癌症发生有密切关系。这三个蛋白激酶在序列上非常相似,尤其是C端的高度保守催化区域,人的A,B有71%的同源性。激酶区含有XRXTXXCGTX的激活环,是Aurora家族特异的保守残基。人Aurora-A和B激酶区的晶体结构已被解析,三维结构显示这两个激酶的功能域高度保守。然而,三个Aurora激酶在N端调节区的长度和序列相差较多,长度分别是128、64以及7个氨基酸,N端只在起始处的几个氨基酸是保守的,其余部分几乎没有序列保守性,这提示三个激酶作用的方式和底物可能有所不同。 尽管大量的研究结果已使人们对Aurora激酶的功能有了较为深入的了解。已知它们参与染色体分离和细胞分裂的多个事件,如中心体形成,纺锤体组装、染色体凝集,动粒-微管接触、纺锤体检验点以及胞质分裂。Aurora激酶表达异常可影响纺锤体组装、检验点功能以及细胞分裂,导致染色体异常分离或者伴随中心体扩增的多倍体细胞。然而迄今为止,这些激酶在体内的作用方式,仍然知之甚少。阐明这类激酶发挥功能的作用方式和调节机制,不仅可以加深对其生理功能的理解,而且有利于利用其独特的调节机制来筛选抗肿瘤的新型小分子药物。本研究以其中最具代表性的Aurora-A为研究对象,深入探讨了它的激酶活性调节机制。 Aurora-A激酶由403个氨基酸组成,包括N端调节区和C端催化区。Aurora-A的N端调节区(1-128aa),包含Aurora box1(3-42aa)和Aurora box2(44-63aa),因为二者只相隔两个氨基酸,一般将二者合称为Aurora Box。我们首先研究了Aurora box是否对激酶区有调节作用。发现在293T细胞中重组表达的人Aurora-A的C端激酶区磷酸化通用底物MBP的活性明显高于全长Aurora-A蛋白约4倍。而且重组表达的N端区多肽能够抑制C端的激酶活性,但对全长Aurora-A更多还原

【Abstract】 The Aurora serine/threonine kinases are currently one of the most intensely studied families of protein kinases, they regulate mitosis and meiosis in all eukaryotes and increasing evidence links these kinase to oncogenesis. Aurora kinases play critical roles in chromosome segregation and cell division. They are implicated in the centrosome cycle, spindle assembly, chromosome condensation, microtubule-kinetochore attachment, the spindle checkpoint and cytokinesis.Aurora kinases are regulated through phosphorylation, the binding of specific partners and ubiquitin-dependent proteolysis.Several Aurora substrates have been identified and their roles are being elucidated. The deregulation of Aurora kinases impairs spindle assembly, checkpoint function and cell division, causing missegregation of individual chromosomes or polyploidization accompanied by centrosome amplification. Aurora kinases are frequently overexpressed in cancers and the identification of Aurora A as a cancer-susceptibility gene provides a strong link between mitotic errors and carcinogenesis.All members of this kinase subfamily possess two distinct domains, a highly conserved C terminal catalytic domain and an N terminal non-catalytic extension that varies in size and sequence. The most conserved motif that can be found is located at the very beginning of the N-terminal domain, the length of this N-terminal domain varies from 7 residues to 162 residues, and seems to be responsible for the specificity of each kinase.Aurora-A plays an important role in centrosome maturation and spindle assemble. Aurora-A is localized at the spindle poles from prophase to telophase, and overexpression results in abnormal centrosome ampilification and cellular transformation in mammalian cells. The AURKA lies within a region of human chromosome 20q13 that is amplified in many forms of cancer, it encodes Aurora-A kinase consisting of 403 aa.The N terminal of Aurora-A contains Aurora boxl (3-42aa) and Aurora box2 (44-63asa), which called Aurora box.The non-catalytic domains of protein kinases fulfil at least two functions in vivo: to regulate the kinase ativity and to localize the protein.It is known that many protein kinases including tyromine kinase Src, MTK1 and ser/thr kinase such as Plk1, can be constitutively activated by eliminating their N-terminal sequences. Thus, their更多还原