ATM与辐射激活的磷酸化P53、P21蛋白的相互作用  

Interaction between ATM and Radiation-activated Phosphorylation of P53 and P21

作　　者：罗加林[1] 曹建平[2] 朱巍[2] 冯爽[2] 盛方军[2] 朱财英[2] 郑斯英[2] 

机构地区：[1]浙江省肿瘤医院放疗科,浙江杭州310022 [2]苏州大学放射医学与公共卫生学院放射生物学教研室,江苏苏州215007

出　　处：《癌症》2005年第9期1059-1063,共5页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30170288);江苏省高校自然科学基金重点项目(No.04KJA180121);江苏省2004年度研究生创新计划项目(No.xm04-67);苏州大学医学发展基金重点项目(No.EE126032)~~

摘　　要：背景与目的:ATM基因属于PI-3K激酶家族成员,其编码蛋白具有调控DNA修复过程和调整细胞周期关卡的功能。毛细血管扩张性共济失调症(ataxia-telangiectasia,AT)患者AT细胞中ATM基因的突变导致了辐射诱发的P53、P21磷酸化缺失,说明辐射激活ATM基因可调控P53、P21的磷酸化。本实验利用免疫共沉淀及Westernblot技术来研究辐射激活的ATM基因与p53的关系,并观察ATM基因是否不通过P53而直接调控P21的磷酸化。方法:利用电穿孔技术将含有ATM基因cDNA的真核表达载体pEBS7-YZ5转染到AT细胞中,用潮霉素筛选以获得稳定表达细胞株,RT-PCR检测ATMcDNA的转录以进一步验证;在ATM稳定表达的AT细胞中,利用免疫共沉淀及Westernblot技术研究ATM基因与p53基因的相互关系;以K562细胞(p53突变)为p53突变细胞模型,研究ATM是否直接磷酸化P21。结果:pEBS7-YZ5成功转进AT细胞,RT-PCR检测到ATMcDNA片段;ATM稳定表达的AT细胞株在电离辐射诱导下,P53被磷酸化,免疫共沉淀显示ATM与P53相互作用;K562细胞经60Coγ射线照射后,P21被磷酸化,ATM抗体免疫共沉淀物中检测到P21蛋白的存在。结论:细胞遭受电离辐射作用后所激活的ATM激酶,可通过磷酸化P53继而活化细胞周期检控点P21蛋白,也可在电离辐射导致DNA损伤早期直接磷酸化P21蛋白,来启动DNA修复机制。BACKGROUND ＆ OBJECTIVE： ATM gene is a member of PI0-3K kinase family. ATM protein is capable of controlling DNA repair process and cell cycle checkpoint, In AT cells from ataxia-telangiectasia （AT） patients, ATM gene mutation leads to the deficiency of ionizing radiationactivated phosphoryiation of P53 and P21. It shows ATM gene could mediate the phosphorylation of P53 and P21. This study was to explore the interaction between ATM and P53, and to observe whether ATM directly medicates the phosphoryiation of P21 in a P53-independent way. METHODS： pEBS7-YZ5 vector containing ATM cDNA was transfected into AT cells by electroperforation. The cells expressing ATM protein stably were screened with hygromycin, and identified by reverse transcription-polymerase chain reaction （RT-PCR）. The interaction between ATM and P53 in pEBS7-YZ5-AT cells was assessed by co-immunoprecipitation and Western blot. K562 cells served as a P53 mutation cell model to study whether ATM could interact with the phosphoryiation of P21. RESULTS： pEBS7-YZ5 was transfected into AT cells successfully, RT-PCR detected fragment of ATM cDNA. After exposed to ionizing radiation, P53 of pEBS7-YZ5-AT cells was phosphorylated, and immunoprecipitation showed interaction between ATM and P53; P21 of K562 cells was phosphorylated, P21 protein was detected in the immunoprecipitation of ATM antibody-complex. CONCLUSION： lonizing radiation-activated ATM kinase could interact with the phosphorylation of P53 and P21 in both P53 wild type and mutant type cells.

关 键 词：ATM P53 P21 AT细胞 K562细胞 电离辐射 磷酸化/辐射效应 DNA损伤/辐射效应 

分 类 号：R744.7[医药卫生—神经病学与精神病学]