Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a ro le in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domai n (Src homology 3) binds proteins at specific, proline-rich sequences, whil e the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequence s. Inhibition of Src SH3 and SH2 domain function is of potential therapeuti c value because of their importance in signaling pathways involved in disea se states, We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous mea surement of compounds binding to each domain in assays based on the techniq ue of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequen ce from the rat protein pl30(CAS) which has been reported to bind simultane ously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, w e confirm that sequences from pl30CAS can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an inter nal control to identify substances which inhibit SH3 and SH2 binding via no nspecific mechanisms. (C) 1999 Academic Press.