Identification of a Src SH3 domain binding motif by screening a random phage display library.
A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.
Cheadle
C
C
Department of Molecular Biology, Rhône-Poulenc Rorer Central Research, Collegeville, Pennsylvania 19426.
Ivashchenko
Y
Y
South
V
V
Searfoss
G H
GH
French
S
S
Howk
R
R
Ricca
G A
GA
Jaye
M
M
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015698:Genomic Library; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015688:Oncogene Protein pp60(v-src); D017868:Cyclic AMP-Dependent Protein Kinases