Proteomic and structural analysis of the Neuronal Src SH3 domain

The ubiquitous non-receptor tyrosine kinase C-Src functions in proliferation, migration and differentiation, and is well characterised in terms of the ligands, substrates and signalling pathways driving these functions. A neuronal-specific splice variant of C-Src, N1-Src, is formed by the insertion of six residues (RKVDVR) in the n-Src loop of the substrate binding SH3 domain. N1-Src is evolutionarily conserved in vertebrates and is expressed in both the developing and adult brain, where it is implicated in neuronal development, differentiation and morphology. However, the ligands and substrates through which N1-Src carries out these functions are unknown. This study aimed to assess the structural impact of the N1-Src insertion, and its effects upon the interactome and phosphoproteome of N1-Src. The ligand and substrate specificity of C- and N1-Src were compared via mass spectrometry analysis of GST-SH3 domain pull-downs and a novel in vitro whole cell lysate kinase assay in a developmental neuronal lysate. Thirty three N1-Src SH3 domain ligands were identified, in comparison to the 176 C-Src ligands, suggesting that N1-Src functions as a tailored isoform in neurons. Furthermore, N1-Src had 239 in vitro substrates, with the phosphorylation sites mapped in over 70 % of the proteins. The ligands and substrates of N1-Src enriched for Gene Ontology terms spanning neuronal development, differentiation, morphology and cytoskeletal regulation, in-line with its cellular functions. A number of the C- and N1-Src SH3 domain ligands were also in vitro substrates, thus providing strong candidates for follow up studies. Interestingly, the C- and N1-Src SH3 domains displayed differential peptide ligand binding profiles via nuclear magnetic resonance spectroscopy. Furthermore, changes to the N1-Src SH3 domain were identified that might explain its tailored ligand binding, and importantly, N1-Src’s increased catalytic activity. Taken together, these proteomic and structural studies have provided a foundation for the structural and in vivo molecular characterisation of N1-Src.