摘要
DNA-PK复合物由Ku蛋白和DNA依赖蛋白激酶催化亚基（DNA-PKcs）组成,DNA-PKcs属于PI3K相关激酶家族成员.我们前期工作发现,DNA-PKcs沉默后,c-Myc的稳定性下降,且二者存在相互作用.为进一步确定c-Myc蛋白与DNA-PKcs相互作用位点,本研究利用原核表达系统活动了c-Myc及其截短体蛋白,利用GSTpull-down技术结合Western印迹法,发现c-Myc蛋白294-370位氨基酸与DNA-PKcs存在相互作用.在细胞内表达GFP-c-Myc各截短体蛋白,发现294-370位氨基酸是c-Myc蛋白降解必需的.利用免疫荧光技术,发现DNA-PKcs与c-Myc蛋白有相同的细胞亚定位,进一步表明两者在生物学功能上具有相关性.有文献报道294-370位氨基酸是乙酰转移酶p300的底物,此位点的乙酰化导致c-Myc的降解.本实验结果提示,c-Myc蛋白的294-370位氨基酸与DNA-PKcs结合,可能阻止了乙酰转移酶p300的结合,从而达到提高c-Myc蛋白稳定性的作用.
DNA-dependent protein kinase （DNA-PK） is an abundant protein complex, comprising the heterodimer of ku protein and a large 450 kD catalytic subunit termed DNA-PKcs （DNA-PK catalytic subunit）. DNA-PKcs is a member of the phosphatidyl inositol 3-kinase （PI 3K）-related kinase subfamily and has been intensively investigated for its roles in DNA non-homologous end-joining （NHEJ）. Mutation defective in DNA-PKes is highly radiation sensitive. DNA-PKcs has been reported to be highly expressed in many tumor tissues, raising the possibility that it will have additional functions. We previously reported that the c-Myc protein level decreased in DNA-PKcs silencing cells, and one of the mechanisms due to the interaction between DNA-PKcs and c-Myc. However, the significance of the interaction hasn＇t been addressed. This study is initiated to determine the binding site between c-Myc and DNA-PKcs to unveil the regulation role of DNA-PKcs in the activity of c-Myc. The binding site of c-Myc with DNA-PKcs is screened by GST pull-down followed Western blotting detection. Binding site is mapped within the domain of 294 - 370 in the carboxyl terminus of c-Myc in vitro. Deletion of this domain increases the stability of c-Myc protein in vivo. It has been reported that the 294 - 370 domain of c-Myc is a substrate of acetyl transferases p300, the interaction between c-Myc and DNA-PKcs might prevent the acetylation of c-Myc at the 294 - 370 domain which decreases the stability of c-Myc. The eoloealization of c-Myc and DNA-PKcs in nuclear of HeLa cells further confirms the interaction. These results help to define a novel pathway by which the stability of c-Myc oneoprotein is regulated.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2009年第1期30-36,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究发展规划(973计划
No.2007CB914603)
国家自然科学基金(No.30371232
No.30500267)资助~~
