Regulation of a neuronal splice variant of Enabled homolog by N1-Src kinase in neurite outgrowth

The astounding complexity of the central nervous system arises from the precise guidance
of axons to their synaptic targets. Aberrant axon guidance is implicated in
neurodevelopmental disorders such as autism. At the distal end of the guiding axon, the
growth cone receives signals that are transduced to the actin cytoskeleton. Principal players
in these transduction pathways are non-receptor protein tyrosine kinases. One such kinase
is N1-Src, a neural isoform of the ubiquitous c-Src, which is enriched in growth cone
membranes and has altered substrate specificity from c-Src. The targets of N1-Src are
poorly understood, but a preliminary substrate screen of N1-Src revealed an exciting
potential candidate, a neural isoform of an actin regulator, N-Enah, with three putative
tyrosine phosphorylation sites. Bioinformatic analysis demonstrated that N1-Src and N-Enah
co-evolved in early vertebrate evolution. Interestingly, the Drosophila homologue of Enah,
Ena, is tyrosine phosphorylated by Abl in a glutamine (Q)-rich domain that is not conserved
in mammals. Ena tyrosine phosphorylation regulates protein-protein interactions necessary
for the regulation of the actin cytoskeleton. In this project, it was hypothesised that the
N-Enah insert and N1-Src function similarly to the Q-rich domain of Ena and Abl in neuronal
differentiation. To investigate the function of N-Enah tyrosine phosphorylation, a phosphonull
(6F) mutant of N-Enah was cloned into a Green Fluorescent (GFP)-expression plasmid and
transfected into neuroblastoma cells with or without FLAG-tagged N1-Src. Compared to the
wild type N-Enah, the 6F mutant could not be detected by immunoblotting with a
phosphotyrosine antibody, was more stably associated with the cytoskeleton following
subcellular fractionation, and inhibited cAMP-dependent neurite outgrowth. Taken together,
these data support the idea that tyrosine phosphorylation of N-Enah regulates the interplay
of protein-protein interactions necessary for actin polymerisation in neuronal differentiation.
Further experiments are needed to identify the key players that are regulated by this
conserved signalling mechanism.