Regulation of the Ras-GTPase activating protein neurofibromin by C-tail phosphorylation: implications for protein kinase C/Ras/extracellular signal-regulated kinase 1/2 pathway signaling and neuronal differentiation.

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase-activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH-SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Surprisingly, we observed that, among other PKC-dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C-tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA-induced differentiation of SH-SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C-tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC-dependent, sustained C-tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.