TI - Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells . AB - The proto oncogene product c-src is a tyrosine-specific kinase with a still unresolved cellular function . High levels of c-src in neurons and the existence of a neuronal c-src variant , pp60c-srcN , suggest participation in the progress or maintenance of the differentiated phenotype of neurons . We have previously reported that phorbol esters , e.g. , 12-O-tetradecanoylphorbol-13-acetate ( TPA ) , stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation , as monitored by morphological , biochemical , and functional differentiation markers . In this report , we describe activation of the pp60src ( c-src and pp60c-srcN ) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA . This phenomenon coincides in time with neurite outgrowth , formation of growth cone-like structures , and an increase of GAP43 mRNA expression , which are the earliest indications of neuronal differentiation in these cells . The highest specific src kinase activity ( a three - to fourfold increase 4 days after induction ) was noted in cells treated with 16 nM TPA ; this concentration is optimal for development of the TPA -induced neuronal phenotype . During differentiation , there was no alteration in the 1 : 1 ratio of c-src to pp60c-srcN found in untreated SH-SY5Y cells . V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells , mainly because of an intense serine 12 phosphorylation . Tyrosine 416 phosphorylation was not detectable in either cell type , and no change during differentiation in tyrosine 527 phosphorylation was observed .