Amer1/WTX与APC相互作用的新结合位点鉴定

Identification of a New Binding Site in Amer1 (365-375) Associated with APC-ARM Domain

Amer1(APC membrane recruitment protein 1)又称为WTX(Wilms’tumor X),是首个发现位于X染色体上的抑癌基因.由于其定位的特殊性,Amer1近年来成为研究的热点之一.研究表明,Amer1作为骨架蛋白在细胞内与多种蛋白(APC,β-catenin,Axin等)直接结合,在Wnt信号通路中发挥着重要功能.据报道Amer1/WTX含有3个肿瘤抑制蛋白质APC(adenomatous polyposis coli,APC)的结合位点,对于APC在细胞膜上的定位过程中发挥着重要的作用.但是,通过序列比对发现,Amer1可能存在第4个被忽略的APC的结合位点,定位于A1和A2之间.为了验证该片段能否与APC结合,分别构建了GST-Amer1(365-375)和His-APC(407-775)两种重组蛋白.通过GSTpull down,证明了这两个片段存在相互作用,并进一步通过ITC(isothermal titration calorimetry)实验测定了两者结合的亲和力.本研究结果不仅在体外证实了Amer1第4个APC结合位点的存在,也为APC和Amer1/WTX复合物的结构和功能的研究打下了良好的基础.

Amerl （APC membrane recruitment protein 1） gene, also known as tumor suppressor WTX （Wilms＇ tumor X） gene, is the first discovered tumor suppressor gene located in the X chromosome. Due to its special location, Amerl is of particular interest for researchers recently. Functional study shows that Amerl plays as an important scaffold protein binding with several proteins （APC, β-catenin, Axin, etc）, which are involved in the Wnt signaling pathway. Especially, it is reported that Amerl is associated with APC membrane location, and it contains three binding sites for APC that share no obvious sequence similarity to each other. However using the method of homologous sequence alignment, we found a potential new APC binding site （365-375） located between A1 and A2, which may be ignored in the previous study. To prove this hypothesis, two recombinant proteins GST-Amerl 365-375 and His- APC 407-775 were cloned, expressed and purified, respectively. By GST-pull down method, significant binding of the two proteins were observed. The affinity of them was also further measured and calculated by isothermal titration calorimetry （ITC） experiment. These results not only prove the existence of the fourth APC binding site in Amerl in vitro, but also provide a solid basis for further structural andfunctional study of Amerl/APC complex.