Molecular mechanism of G2/M Progression by protein kinases

1.（1）制备抗Aurora-A的单克隆抗体，并测定Aurora-A在人细胞中的定位。(2)我们发现人Aurora-A降解依赖于hCdh 1通过APC/C-ubiquitin-proteasome途径。(3)我们确定了人Aurora-B的底物特异性。我们发现，这种酶是一种丝氨酸导向的激酶，可以磷酸化组蛋白H3的丝氨酸10和28在体外。我们提出了单克隆抗体对有丝分裂特异性的自磷酸化位点的极光-A的T-环，并确定了极光-A在人类细胞中激活的时间和位置。(4)我们确定Aurora-B相关蛋白磷酸酶作为激酶激活的负调节因子。(5)我们研制了一种能识别Aurora家族所有成员的单克隆抗体。(6)我们开发了一种Aurora-A反义寡核苷酸方法，并阐明了Aurora-A反义寡核苷酸处理的Aurora-A耗竭导致细胞周期在前中期显著停滞。(7)我们发现，MBD 3，组蛋白去乙酰化酶NuRD复合物的一个组成部分，是在体内磷酸化的晚期G2和早期M期。此外，我们发现Aurora-A在体外磷酸化MBD 3，在体内与MBD 3物理缔合，并在早期M期与MBD 3共定位于中心体。(8)我们构建了一个人稳定的细胞系，其中Aurora-A，组蛋白H3和importina差异表达为融合绿色，青色和红色荧光蛋白。利用先进的时间推移图像分析系统，对其在有丝分裂活细胞中的分子行为进行了广泛的分析. SNK（1）我们制备了抗SNK的单克隆抗体，发现这些抗体是早期发现甲状腺癌的有用的诊断工具。

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.