IDENTIFICATION OF A SRC SH3 DOMAIN BINDING MOTIF BY SCREENING A RANDOM PHAGE DISPLAY LIBRARY

A phage display library was constructed in the filamentous bacteriopha ge fuse5. The library was made by inserting a degenerate oligonucleoti de which encodes 15 variable amino acids into the NH,(2)-terminal regi on of the phage gene III protein. This library containing over 10(7) d ifferent phage, was screened with a glutathione S-transferase (GST) fu sion protein containing the Src homology 3 (Src SH3) domain and a prot ein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proli ne-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifi cally bind to the GST/PHA/Src SH3 protein but not to GST/PKA only. A c omparison of the inferred amino acid sequence of the different phage c lones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity s creen of a lambda-lox mouse embryo cDNA library using a P-32-labeled S rc SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in pr eparation). Peptides based upon the 7-amino acid SH3 binding domain co re motif displayed strong binding to both the Src and to the Fyn SH3 d o mains, but failed to bind to the SH3 domain of p21Ras-GTPase-activat ing protein (Ras GAP) and other proteins. We anticipate that further s creening of the phage display library will be a useful tool for the ra pid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the spec ificity of interactions among the diverse proteins containing SH3 doma ins and those containing SH3 binding motifs.