c-Myc蛋白与DNA-PKcs作用位点的鉴定

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-Kcs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GST pull-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.

Identification of Interaction Site of c-Myc with DNA-PKcs

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-PKcs 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 colocalization 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 oncoprotein is regulated.