鉴定人甲基化CpG结合结构域蛋白4为蛋白激酶X的底物(英文)

Identification of Human Methyl-CpG Binding Domain Protein(MBD) 4 as a Substrate of Protein Kinase X

人蛋白激酶X(PrKX)是由X染色体编码的一种cAMP依赖性蛋白激酶,但是到目前为止已鉴定到的PrKX底物还很少.为了鉴定蛋白激酶X的底物,我们以蛋白激酶X为诱饵进行了酵母双杂交实验,结果发现甲基化CpG结合结构域蛋白4(MBD4)与PrKX在酵母细胞内相互作用较强.GST融合蛋白沉降和细胞内蛋白质的免疫共沉淀证实PrKX与MBD4之间确实存在相互作用.进一步研究表明,大肠杆菌中表达的重组MBD4在体外可以被PrKX磷酸化,而且MBD4蛋白的磷酸化能明显增强它在体外与甲基化DNA探针的结合活性.

Human protein kinase X （PrKX） is a cAMP-dependent protein kinase encoded by X chromosome. However, few substrates have been identified so far. To identify the substrates of PrKX, we employed a yeast two-hybrid screen using PrKX as the bait. The human protein MBD4 was isolated as a strong interaction partner. The stability and specificity of interaction were confirmed by in vitro pull-down experiments and immunoprecipitation of complexes assembled in vivo. Subsequently, we showed that the hMBD4 protein expressed in E. coli could be phosphorylated by PrKX. Phosphorylation of hMBD4 by PrKX modulated the binding activity ofhMBD4 to fully methylated DNA. These results indicate that MBD4 is a substrate for PrKX.