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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.
The DNA-PK complex consists of Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the PI3K-related kinase family. Our previous work found that the stability of c-Myc after DNA-PKcs silencing Decreased and interacted with each other.In order to further confirm the interaction between c-Myc and DNA-PKcs, c-Myc and its truncated protein were activated by prokaryotic expression system, and GSTpull-down combined with Western Western blotting showed that the amino acids 294-370 of c-Myc interacted with DNA-PKcs.The expression of GFP-c-Myc truncated protein in cells was found to be essential for the degradation of c-Myc from amino acids 294 to 370 The results of immunofluorescence showed that DNA-PKcs had the same cellular sub-localization with c-Myc protein, which further indicated that the two genes were correlated in biological function.It has been reported in the literature that the amino acids 294-370 are the base of acetyltransferase p300 Acetylation of this site leads to the degradation of c-Myc.The results of this experiment suggest that binding of amino acids 294-370 of c-Myc protein to DNA-PKcs may prevent the binding of acetyltransferase p300 and thus increase c The role of Myc protein stability.