Figure 1: Identification of a novel ligand consensus sequence for the N1-Src SH3 domain.
(a) Schematic of the kinase assay used to validate the effect of potential SH3 domain ligands on the phosphorylation of an ideal tyrosine motif (Y in bold) located upstream of a 10 aa linker and 12 aa phage peptide. (b) GST-Y-PDN1–6 substrates at 0.3, 0.9, 2.8 or 8.3 μM were phosphorylated in vitro with 5 nM C- or N1-Src at 30 °C and the phosphorylation detected by anti-phosphotyrosine immunoblot. Immunoblots are representative of three independent experiments. (c) Alignment of the phage display peptides ranked by N1-Src selectivity derived from results in (b). Shaded residues represent the common consensus sequence. (d) Kinetic measurements for GST-Y (Y), GST-Y-PDN1 or GST-Y-PDN2 substrates (see a for structure) in the presence of 5 nM C- or N1-Src. Data were plotted as mean Km (μM) ± SEM (n = 3). *P < 0.05 by ANOVA and post-hoc Tukey test. (e) GST-Y-PDN1 substrates with the indicated point mutations (see insert) at 8.3 μM were phosphorylated by 5 nM N1-Src and the extent of phosphorylation was measured by densitometry of phosphotyrosine immunoreactivity. Data were plotted as arbitrary units (±SEM, n = 3), normalised to the wild-type (WT) PDN1 peptide. **P < 0.01; ***P < 0.001 by ANOVA and post-hoc Tukey test.
