DISTINCT LIGAND PREFERENCES OF SRC HOMOLOGY-3 DOMAINS FROM SRC, YES, ABL, CORTACTIN, P53BP2, PLC-GAMMA, CRK, AND GRB2

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in sig nal transduction. These domains have been shown to mediate protein-pro tein interactions by binding short proline-rich regions in ligand prot eins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein inter actions remain poorly defined. We have used a phage-displayed library of peptides of the form X(6)PXXPX(6) to identify ligands for eight dif ferent SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characterist ics. Specifically, we have found that the Src SH3 domain selects pepti des sharing the consensus motif LXXRPLPX Psi P, whereas Yes SH3 select s Psi XXRPLPXLP, Abl SH3 selects PPX Theta XPPP Psi P, Cortactin SH3 s elects +PP Psi PXKPXWL, p53bp2 SH3 selects RPX Psi P Psi R+SXP, PLC ga mma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects Psi P Psi LP P si K, and Grb2 N-terminal SH3 selects +Theta DXPLPXLP (where Psi, Thet a, and + represent aliphatic, aromatic, and basic residues, respective ly). Furthermore, we have compared the binding of phage expressing pep tides related to each consensus motif to a panel of 12 SH3 domains. Re sults from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains t o discern subtle differences in the primary structure of potential lig ands. Finally, we have found that most known SH3-binding proteins cont ain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.