Role of protein tyrosine phosphorylation in the NGF response

The role of protein tyrosine phosphorylation in the response of PC12 cells to NGF was investigated by using a variety of agents which affect NGF-induced neurite outgrowth. K-252a, a kinase inhibitor, was previously found to selectively inhibit many of the actions of NGF on PC12 cells. In the present study, it was shown to inhibit NGF-induced protein tyrosine phosphorylation. However, sphingosine, an inhibitor of protein kinase C and NGF-induced differentiation of PC 12 cells, did not alter the phosphorylation of proteins on tyrosine stimulated by NGF. Disruption of either actin microfilaments or microtubules also had no effect on NGF-induced protein tyrosine phosphorylation in PC12 cells. The effect of vanadate, an inhibitor of phosphotyrosyl phosphatases, on the differentiation of PC12 cells was also examined. Vanadate did not promote neurite outgrowth but did stimulate protein tyrosine phosphorylation. Taken together, these results suggest that protein tyrosine phosphorylation is one of the first events in the NGF pathway in PC12 cells but alone is not sufficient to induce morphological differentiation. Finally, the distribution of phosphotyrosine-containing proteins in untreated and NGF-treated cells was examined by immunofluorescence microscopy. The distribution of these proteins was altered by treatment of the cells with NGF and appeared to correlate with the distribution of actin filaments, particularly in growth cones.