EXAMINING THE SPECIFICITY OF SRC HOMOLOGY-3 DOMAIN-LIGAND INTERACTIONS WITH ALKALINE-PHOSPHATASE FUSION PROTEINS

Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk , p53BP2, and Src, were fused to the N-terminus of Escherichia coli al kaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interaction s on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and sp ecific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo lambda cDNA expression library and resulted in the cloning of p53BP2 and seve ral known proteins with SH3 domains similar to that of Cortactin, resp ectively. In addition, the similar to 60-amino-acid-long SH3 domains o f Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide Ligands in micr otiter plates and proteins containing proline-rich regions in screens of a lambda cDNA expression Library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for e xamining the specificity of SH3 domain-ligand interactions, identifyin g potentially interacting proteins, and establishing high-throughput s creens of combinatorial chemical libraries. (C) 1997 Academic Press.