【摘要】 目的筛选与PTEN结合的含PDZ结构域的蛋白,并利用蛋白质组学方法对筛选出的蛋白质的相互作用予以鉴定。方法利用PDZ蛋白质芯片筛选PTEN的结合蛋白。对所发现的PTEN与NHERF-1的相互作用,应用GST融合蛋白沉降和免疫共沉淀实验等多种方法予以鉴定。结果发现了几种新的与PTEN结合的含PDZ结构域的蛋白质。并对PTEN/NHERF-1的相互作用进行了进一步研究,发现PTEN的羧基端可与NHERF-1的第一个PDZ结构域(PDZ1)结合。将PTEN羧基最末端的4个氨基酸(I-T-K-V)中任何一个突变为丙氨酸,都明显抑制PTEN与NHERF-1的结合。免疫共沉淀结果显示,在细胞内完整的蛋白质分子PTEN与NHERF-1也可以结合。结论PTEN与NHERF-1之间存在相互作用,该相互作用是由PTEN的羧基末端与NHERF-1的PDZ1结构域结合而实现的,PTEN羧基最末端的4个氨基酸对于维持它们之间的结合有十分重要的作用。更多还原

【Abstract】 Objective To identify PDZ domain containing proteins interacting with PTEN and its characterization with NHERF-1 by proteomic analysis. Methods The interactions between PTEN and PDZ domain containing proteins were screened with PDZ protein array, and the novel one was then identified with GST pull-down and co-immunoprecipitation assay. Results Using a PDZ protein array, we proved PTEN binding with NHERF-1. The interaction of PTEN and NHERF-1 was further characterized by GST pull down assay, and we demonstrated that PTEN associated with NHERF-1 via the binding of PTEN carboxyl-terminal with the PDZ domain 1 (PDZ1) of NHERF-1. The last four amino acids (I-T-K-V) of the PTEN were the key determinants of this interaction as mutation of any of the four amino acids to alanine resulted in markedly reducing association of PTEN with NHERF-1. In addition, the full-length of PTEN robustly associated with NHERF-1 was also determined by co-immunoprecipitation experiment in cos-7 cells. Conclusion PTEN/NHERF-1 association was mediated via the binding of PTEN carboxyl-terminal]with the PDZ1 of NHERF-1, and the last four amino acids of the PTEN carboxyl-terminal were important for PTEN/NHERF-1 interaction.更多还原