PROLINE-RICH SEQUENCES THAT BIND TO SRC HOMOLOGY-3 DOMAINS WITH INDIVIDUAL SPECIFICITIES

To study the binding specificity of Src homology 3 (SH3) domains, we h ave screened a mouse embryonic expression library for peptide fragment s that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhi bit differential binding specificity to various SH3 domains. Src-SH3-s pecific binding uses a sequence of 7 aa of the consensus RPLPXXP, in w hich the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3, in contrast; a quite different p roline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn , and Hck SH3 domains, but not to the Src SH3. Specific binding of the AM SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indis pensable for binding to all tested SH3 domains occurs at the C terminu s. Another clone contains overlapping yet distinct Src and Abl SH3 bin ding sites, Binding to the SH3 domains is mediated by a common PXXP am ino acid sequence moth present on all ligands, and specificity comes a bout from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.