Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.
To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.
Yokoyama
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N
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794-8661, USA.
Miller
W T
WT
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics* / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity
D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D015688:Oncogene Protein pp60(v-src)