Structure-function studies of the neuronal Src kinases

N1- and N2-Src are neuronal specific splice variants of the ubiquitously expressed tyrosine kinase C-Src. They differ only by short amino acid inserts within their SH3 domains, a region known to confer substrate specificity. Due to their highly identical sequence it has been difficult to attribute specific neuronal functions to each Src enzyme, however, many C-Src SH3 domain substrates do not bind to the N-Src SH3 domains. The limited functional N1-Src data indicate that it is involved in neuronal differentiation. Furthermore, high expression levels of the N-Srcs in childhood neuroblastoma correlate with cases in which the tumour spontaneously differentiates to a harmless neuronal phenotype. In this study, I sought to explain how the amino acid inserts in the N-Src SH3 domains affect substrate specificity and kinase activity and how they might act to drive neuronal differentiation.
I employed a multi-disciplinary approach to investigate the functions of the N Srcs. Studies in heterologous cells revealed a specific role for the N-Srcs in cytoskeletal rearrangement. A sensitive in vitro kinase assay was developed and this showed that the N-Src SH3 domain ligand preferences differ from those of C-Src. A subsequent phage display screen was able to identify a novel consensus sequence for the N1-Src SH3 domain and peptides containing this consensus motif were shown to be highly specific N1-Src inhibitors both in vitro and in cells. Bioinformatic analyses revealed the consensus sequence to be present in many neuronal proteins and identified a number of putative N1-Src substrates. In cultured neurons I identified a specific role for N1-Src acting in the L1-CAM pathway to modulate neurite outgrowth.
The data presented here provide evidence that the inserts in the SH3 domains of the N-Srcs confer significant differences in their substrate preferences and that the functions mediated by the N-Srcs are different to those of C-Src. A role for N1-Src has been identified in the modulation of axon outgrowth in cultured neurons and the putative substrates identified now provide promising targets for the further study of N1-Src function. Future investigations will be able to utilise the data presented here to elucidate how N1-Src regulates the neuronal cytoskeleton, while tools I have developed; including a highly specific N1-Src inhibitor will greatly aid these investigations.