Dot far-western blot analysis of relative binding affinities of the Src homology 3 domains of Efs and its related proteins.
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.
Ohba
T
T
Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Sapporo, Chuo-Ku, 060-8556, Japan.
Ishino
M
M
Aoto
H
H
Sasaki
T
T
D048868:Adaptor Proteins, Signal Transducing; D001345:Autoradiography; D001667:Binding, Competitive; D015153:Blotting, Western / Q000379:methods*; D029563:Cellular Apoptosis Susceptibility Protein; D051418:Focal Adhesion Kinase 2; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D029563:Cellular Apoptosis Susceptibility Protein; C484351:EFS protein, human; C099942:NEDD9 protein, human; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D051076:Proto-Oncogene Proteins c-fyn