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Molecular mechanism of G2/M Progression by protein kinases

蛋白激酶G2/M进展的分子机制

基本信息

批准号：
12470036
负责人：
URANO Takeshi
金额：
$ 4.29万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

1. Aurora(1) We raised monoclonal antibodies against Aurora-A and determined the location of Aurora-A in human cells.(2) We showed that human Aurora-Adegradation is dependent on hCdh1 through the APC/C-ubiquitin-proteasome pathway.(3) We determined the substrate specificity of human Aurora-B. We found that this enzyme is an arginine-directed kinase that can phosphorylate histone H3 at serines 10 and 28 in vitro. We raised monoclonal antibodies against mitosis-specific autophosphorylation site in the T-loop of Aurora-A and determined the timing and location of Aurora-A activation in human cells.(4) We identified Aurora-B associated protein phosphatases as negative regulators of kinase activation.(5) We raised a monoclonal antibody which recognized all of Aurora family.(6) We developed an Aurora-A antisense oligonucleotide method and elucidated that depletion of Aurora-A by Aurora-A antisense oligonucleotide treatment causes a significant cell cycle arrest at prometaphase.(7) We found that MBD3, a component of the histone deacetylase NuRD complex, is phosphorylated in vivo in the late G2 and early M phases. Moreover, we found that Aurora-A phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase.(8) We constructed a human stable cell-line in which Aurora-A, histone H3 and importina were differentially expressed as fusions to green, cyan, and red fluorescent proteins. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system.2. SNK(1) We raised monoclonal antibodies against SNK and revealed that thses antibodies are useful diagnostic tools to find thyroid cancers at an early stage.

1.Aurora(1)我们制备了针对Aurora-A的单抗，并确定了Aurora-A在人细胞中的位置。(2)我们证明了人Aurora-A的降解依赖于hCDh1通过APC/C-泛素-蛋白酶体途径。(3)我们确定了人Aurora-B的底物特异性。我们在体外发现该酶是一种精氨酸导向的激酶，可以在丝氨酸10和28处磷酸化组蛋白H3。我们提出了针对Aurora-A T环中有丝分裂特异性自磷酸化位点的单抗，并确定了Aurora-A在人类细胞中的激活时间和位置。(4)我们鉴定了Aurora-B相关蛋白磷酸酶是激酶激活的负调节因子。(5)我们提出了一种识别Aurora家族所有成员的单抗。(6)我们建立了Aurora-A反义寡核苷酸方法，并阐明了Aurora-A反义寡核苷酸处理Aurora-A会导致细胞周期在中期显著停滞。(7)我们发现组蛋白去乙酰基酶NuRD复合体中的MBD3，是组蛋白去乙酰化酶NuRD的组成部分在G2晚期和M期早期在体内被磷酸化。此外，我们还发现，Aurora-A在体外磷酸化MBD3，在体内与MBD3物理结合，并在M期早期与MBD3共定位于中心体。(8)我们构建了一个稳定的人细胞系，其中Aurora-A、组蛋白H3和重要性蛋白差异表达为绿色、青色和红色荧光蛋白。利用先进的时间推移图像分析系统对其在有丝分裂细胞中的分子行为进行了广泛的分析。SNK(1)我们提出了抗SNK的单抗，并表明这些抗体是早期发现甲状腺癌的有用的诊断工具。