The search and analysis for essential signaling mediators responding to radiation by proteome techniques.

为了分离和鉴定辐射应答的重要信号介质，我们寻找了调节辐射诱导的DNA损伤与细胞周期阻滞和凋亡性细胞死亡之间的信号通路的蛋白质，并通过蛋白质组技术分析了它们的功能。过去三年取得的成果可概括如下。我们研究了X射线照射胸腺细胞凋亡过程中染色体乘客蛋白（CPP），包括Aurora-B，生存素和INCENP的亚细胞定位的改变。Aurora-B和Survivin在去污剂不溶物中存在，INCENP在辐射后被caspase-3切割，提示CPPs复合物的异常形成导致细胞凋亡和染色体分离错误.通过蛋白质组学分析，我们确定了Aurora-B的新底物--RNA甲基转移酶NSUN 2，并证实Aurora-B在有丝分裂过程中通过丝氨酸139位的磷酸化参与调节RNA甲基转移酶的活性.我们分析了细胞蛋白质从人类白血病Jurkat细胞照射γ射线或紫外线使用二维凝胶电泳（2DE）。在这些蛋白质点中，利用质谱技术通过肽质量指纹图谱鉴定了酸性核糖体蛋白P2（RpP 2），发现RpP 2的去磷酸化可能与辐射诱导Jurkat细胞凋亡有关.对MDM 2-MDMX复合物生物学特性的研究结果表明，MDM 2与MDMX的相互作用代表了p53调控的一种新模式，提示除p53外，Aurora激酶和酸性核糖体蛋白在辐射诱导的信号转导中也起重要作用。

To isolate and identify the essential signaling mediators responding to radiation, we have searched the proteins regulating signaling pathways between radiation-induced DNA damage and cellular consequences such as cell cycle arrest and apoptotic cell death, and analyzed their functions by proteome techniques. The results obtained for the past 3 years can be summarized as follows.1. We have examined alteration of the subcellular localization of chromosome passenger proteins (CPPs) including Aurora-B, survivin and INCENP during apoptosis in X-irradiated thymic cells. Aurora-B and survivin were present in detergent insoluble fraction and INCENP cleaved by caspase-3 after radiation exposure, suggesting that the abnormal formation of CPPs complex lead to the induction of apoptosis and chromosome segregation errors.2. We could identify the RNA methyltransferase NSUN2 as a novel substrate of Aurora-B by proteome analysis, and demonstrated that the Aurora-B participate to regulate the RNA methyltransferase activity via phosphorylation at Ser139 during mitosis.3. We have analyzed cellular proteins from human leukemic Jurkat cells irradiated with gamma-rays or UV using two dimensional gel electrophoresis (2DE). Among intensive protein spots, acidic ribosomal protein P2 (RpP2) was identified by peptide mass fingerprinting using a mass spectrometry and found that the dephosphorylation of RpP2 may be associated with induction of apoptosis in Jurkat cells by radiation.4. The results of experiments concerning biological properties of MDM2-MDMX complex suggest that the interaction between MDM2 and MDMX represents a novel mode of p53 regulation.These results suggest that Aurora kinases and acidic ribosomal proteins in addition to p53 play an important role in radiation-induced signal transduction.