Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies
Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies
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DOI：
10.1038/sj.onc.1209754
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发表时间：
2006-12-01
期刊：
影响因子：
8
通讯作者：
Urano, T.
中科院分区：
文献类型：
--
作者：
Ohashi, S.;Sakashita, G.;Urano, T.
哺乳动物 Aurora-A 与丝氨酸/苏氨酸蛋白激酶相关，最初因其与酿酒酵母 IpI1p 和果蝇 aurora 的密切同源性而被识别，而这两种蛋白是有丝分裂事件协调的关键调节因子。 Aurora-A 的蛋白质水平、有丝分裂期间的峰值激酶活性及其激活均归因于磷酸化。在这里，我们证明这种酶是一种精氨酸导向的激酶和 de。其底物特异性。我们还发现，激活环内的 Thr288 是体外激活磷酸化事件的关键残基，并且它在体内时空限制于复制中心体和靠近两极的纺锤体微管上有丝分裂时的短暂窗口。免疫耗竭分析表明，除了自磷酸化之外，哺乳动物细胞中可能还存在 Aurora-A 的上游激酶。此外，人激活的 Aurora-A 与在 Thr320 上被负磷酸化的负调节蛋白丝氨酸/苏氨酸磷酸酶 1 (PP1) 形成复合物。有趣的是，磷酸化特异性 Aurora-A 单克隆抗体在体外抑制 Aurora-A 激酶活性，提供了进一步探索的治疗途径。
Mammalian Aurora- A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae IpI1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and de. ne its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase( s) of Aurora- A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora- A forms complexes with the negative regulator protein serine/ threonine phosphatase type 1 ( PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho- specific Aurora- A monoclonal antibodies restrain Aurora- A kinase activity in vitro, providing further therapeutic avenues to explore.