Ohba T, Ishino M, Aoto H, Sasaki T
Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Sapporo, Chuo-Ku, 060-8556, Japan.
Anal Biochem. 1998 Sep 10;262(2):185-92. doi: 10.1006/abio.1998.2772.
The Src homology 3 (SH3) domains are a modular structure of about 60 amino acid residues found in many proteins important in signal transduction. Each SH3 domain has a binding specificity to sequences containing a PXXP motif in ligand proteins. We found that a focal adhesion kinase (FAK)-related protein, cell adhesion kinase beta (CAKbeta), was bound in vitro by the SH3 domain of embryonal Fyn-associated substrate (Efs), a docking protein structurally related to p130Cas (Cas) and HEF1. Here, we employed a dot far-Western blotting technique to evaluate the affinity and specificity of the binding by the SH3 domains of Efs and its related proteins. The SH3 domains and their ligands were prepared as glutathione S-transferase fusion proteins, and one of the binding components was immobilized on membranes while the other was labeled with 32P to use as a probe. The amount of the bound probe was determined by autoradiography using an imaging plate and a bioimaging analyzer. A competitive binding assay showed that Efs, compared with Cas and HEF1, had a SH3 domain with a lower relative affinity to CAKbeta and FAK and with a preference to interact with FAK rather than CAKbeta. Our assay based on dot far-Western blotting is a simple and sensitive method to evaluate fine differences in the binding affinity of SH3-mediated interactions.
Src同源结构域3（SH3）是一种由约60个氨基酸残基组成的模块化结构，存在于许多在信号转导中起重要作用的蛋白质中。每个SH3结构域对配体蛋白中含有PXXP基序的序列具有结合特异性。我们发现一种粘着斑激酶（FAK）相关蛋白，细胞粘着激酶β（CAKbeta），在体外被胚胎Fyn相关底物（Efs）的SH3结构域结合，Efs是一种在结构上与p130Cas（Cas）和HEF1相关的对接蛋白。在此，我们采用斑点远Western印迹技术来评估Efs及其相关蛋白的SH3结构域结合的亲和力和特异性。将SH3结构域及其配体制备为谷胱甘肽S-转移酶融合蛋白，其中一种结合成分固定在膜上，而另一种用32P标记用作探针。通过使用成像板和生物成像分析仪的放射自显影法测定结合探针的量。竞争性结合试验表明，与Cas和HEF1相比，Efs的SH3结构域对CAKbeta和FAK的相对亲和力较低，且更倾向于与FAK而非CAKbeta相互作用。我们基于斑点远Western印迹的检测方法是一种简单且灵敏的方法，可用于评估SH3介导相互作用的结合亲和力的细微差异。
