Affinity Capillary Electrophoresis: Insights into the Binding of SH3 Domains by Peptides Derived from an SIIS-Binding Protein

Src Homology III (SHB) domains are small (55-?0 amino acids) receptor domains that are found in many signaling proteins and are involved in intracellular signaling events.t2 For example, the SH3 domain in the adaptor protein GRB-2 facilitates the coupling of growth factor receptors to an essential switch in growth-related signaling pathways-the guanine nucleotide binding protein Ras.3 Another example is the SH3 domain in the signaling protein PLC-r;this domain is essential for its subcellular localization at actin microfilaments, where signaling results in PlC-7-mediated cytoskeletal rearrangements.4 Other SH3 domains such as those in the Abl and Src tyrosine kinases have been impiicated in oncogenic pathways.s The molecular details of SH3-mediated signaling, however, remain elusive. Recent evidence suggests that SH3 domains bind to proline-rich peptide motifs found in SH3-binding proteins (3BPs). By screening a lgt1l cDNA expression library with the SH3 domain of Abl fused to glutathione Stransferase (GST), Baltimore and co-workers were able to isolate an SH3-binding protein,3BP1.6 Subsequent filter binding assays using GST-3BP1 and several biotinylated GST-SH3 fusion proteins revealed that BBP1 also binds specifically to the SH3 domain of Src. Colorimetric detection of the 3BP1-SH3 complexes using streptavidin alkaline phosphatase yielded similar intensities for the SH3 domains of Abl and Src, indicating that they have comparable affinities for 3BP1. Other GST-fusion protein probes containing the SH3 domains of neural Src and Crk produced much weaker signals. Similar experiments localized the binding site of 3BP1 for the Abl SH3 domain to a ten amino acid region, APTMPPPLPP.? The