The role of N-Src kinases in neuronal differentiation

The ubiquitous proto-oncogene C-Src has two neuronal splice variants, N1- and N2-Src, which contain 6 and 17 amino acid inserts in their SH3 domains respectively. These inserts are thought to modify SH3 domain binding in a manner that decreases auto-inhibition and changes substrate specificity. Although high levels of neuronal Src expression are associated with neuronal differentiation, both during development and in the developmental cancer neuroblastoma, the functions, molecular mechanisms and specific substrate proteins of neuronal Srcs remain largely uncharacterised.
Employing a highly multidisciplinary approach, this project aimed to characterise the role of N-Src expression in neuronal differentiation. Neuronal Srcs were demonstrated to be highly active in neuroblastoma cell lines, and overexpression can drive significant neuritogenesis in the retinoic acid-resistant cell lines KELLY and SK-N-AS. N2-Src expression was also shown to decrease the expression of Ki67 in SK-N-AS cells, indicating that N2-Src can drive neuroblastoma cells into quiescence.
Using the Xenopus embryo as a model system for neuronal development, the expression pattern of xN1-Src during neurulation was characterised and a novel neuronal splice variant was identified in this species. It was demonstrated that xN1-Src is essential for healthy primary neurogenesis, and that xN1-Src knockdown caused a dramatic locomotive and patterning phenotype in X.tropicalis. Using stable, inducible HeLa cell lines, a phosphoproteomic screen demonstrated significant changes in the phosphotyrosine profile between C- and N2-Src over-expressing cells. Several candidate N2-Src substrates were identified, including paxillin, plakophilin and BCAR1. Bioinformatic analyses of the proteomic data revealed the enrichment of signalling pathways and protein complexes involved in membrane traffic and cell adhesion.
Through these multidisciplinary approaches, the cellular effects of N1- and N2-Src signalling during both neuronal precursor and neuroblastoma differentiation have been characterised. Furthermore, a library of potential N-Src substrates has been generated that provides a framework for future studies.