The Role of N1-Src in Neuronal Development

Protein phosphorylation by tyrosine kinases evolved in multicellular organisms to regulate intracellular signalling pathways associated with proliferation, differentiation and migration. In most tissues, basal protein tyrosine phosphorylation is maintained at low levels, but in the brain, basal tyrosine kinase activity is high and regulates key processes in the developing and mature brain and is dysregulated in neurological disorders. N1-Src is a neuronal splice variant of the ubiquitous proto-oncogene C-Src tyrosine kinase, which differs by a six amino acid insert in its SH3 domain. Since the SH3 domain confers substrate specificity, it is anticipated that both C- and N1-Src will have different substrates and functions. Specifically, N1-Src is highly active in the developing brain and has been implicated in neuronal differentiation. Studies also suggest a role for N1-Src in ion channel regulation, however, the mode of action of N1-Src remains poorly understood. The primary aim of this study was to further clarify the role of N1-Src in both the developing and adult brain. To achieve this, a multidisciplinary approach was adopted, which sought to 1) identify novel N1-Src substrates 2) determine the function of N1-Src in developing neurons and 3) dissect the signalling pathways downstream of N1-Src. Recombinant, active Src kinases were generated to undertake in vitro kinase assays with putative N1-Src substrates. Src-dependent phosphorylation of HCN1, a pacemaker channel identified as an N1-Src interactor in a yeast 2-hybird screen, could not be detected. This result was not conclusive as surprisingly, the assay did not detect Src or PKC phosphorylation of NR2A, an NMDA receptor subunit, previously characterised as a robust Src and PKC substrate. However, a screen of several putative N1-Src SH3 binding peptides revealed some encouraging candidates to pursue as substrates. To address the function of N1-Src in neuronal development, N1-Src was overexpressed or knocked down in cultured hippocampal neurons. Both manipulations were detrimental to neurite outgrowth and neuronal polarization, suggesting that N1-Src activates cytoskeletal remodelling pathways and precise levels of N1-Src are required for normal cellular development in vitro. The molecular mechanism of this phenomenon was investigated in a fibroblast cell line, in which N1-Src overexpression induces neurite-like processes. Using this model, an investigation into the role of N1-Src in RhoA signalling implied that N1-Src does not drive process outgrowth via the inhibition of RhoA, however constitutive activation of RhoA, prevented N1-Src mediated process extension. Preliminary results suggested that N1-Src overexpression enhances RhoA activation, which could form part of a negative feedback loop. Taken together, I have implicated N1-Src in neurite outgrowth, which provides a starting point for understanding the mechanistic role of N1-Src in pathways that dictate neuronal morphology