The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src

•N2-Src is a previously uncharacterised neuronal splice variant of C-Src kinase.•Tyrosine phosphorylation by C-Src is enhanced by SH3 peptide ligands.•Ideal C-Src SH3 ligands do not enhance substrate phosphorylation by N2- or N1-Src kinase.•N2-Src is more active than C- and N1-Src in vitro and in cells.•N2-Src is likely to have alternative substrates in the brain. N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain.