Abstract
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. One of the most prominent and distinguishing features of neuronal differentiation is neurite outgrowth. The mechanism by which NGF causes the cells to elaborate neurites is unknown. This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increase in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosphorylation of these three proteins. In the phosphorylation of cell-free extracts with [gamma-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF promoted outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D-resistant neurites were formed and these were blocked by K-252a, showing that the inhibitor interferes with the transcription-independent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probable explanation is that both transcription-dependent and -independent components are involved in at least one step of the activation of some specific protein kinase(s) that can be suppressed by K-252a.
Highlights
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells
Treatment of PC12h cells with NGF (50 ng/ ml) in culture caused an increase in the incorporation of 32p into the 60,000- and 35,000-D proteins, and a decrease in the 36,500-D protein
The data presented here show that K-252a prevented the effects of NGF on phosphorylation of proteins and neurite outgrowth in PC12h cells
Summary
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGE Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. PC12 cells respond to NGF over a period of days by their biochemical and morphological conversion from chromatfin-like cells to those with a sympathetic neuron-like phenotype that includes neurite outgrowth, electrical excitability, and cessation of cell growth [6, 10]. These actions are initiated by the binding of NGF to its specific receptors on the surface of. The exact mechanism of signal transduction after the interaction of NGF with its receptor is unknown, some kind of second messenger system must be involved [16]
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