Role of tyrosine phosphorylation in NGF-mediated signal transduction in PC12 cells

PC12 cells have been used for studying processes related to neuronal differentiation. Upon stimulation with either FGF or NGF, these cells undergo a series of changes resulting in sympathetic neuron-like cells, while treatment with either insulin or EGF induces proliferation of these cells. Since tyrosine kinases have been implicated in both the proliferative and differentiative response, we have used PC12 cells to assess the role of tyrosine kinases and other signaling molecules in NGF-mediated signal transduction. Oncogenic tyrosine kinases related to Src or the FGF receptor, were able to induce process outgrowth while one related to the EGF receptor, Erb B, was unable to do so. These results suggest that only specific classes of tyrosine kinases are able to couple with the signaling molecules mediating neurite extension. To further assess the role of Src family kinases in NGF signaling, we created a PC12 cell line expressing a temperature sensitive v-src gene (ts:v-src(3)) and compared the pathways used by v-src to those used by NGF for the differentiation of PC12 cells. These studies indicated that NGF and v-src use common pathways in the differentiation of PC12 cells and suggest that c-Src may play a role in NGF signal transduction. Further studies demonstrated that Src is required for both NGF- and FGF-induced process outgrowth. Since Ras has also been shown to be required for NGF-mediated signaling in PC12 cells, we wished to determine where Ras functions relative to Src. Microinjection of the GSras1 cells with antibodies against Src was unable to inhibit ras-induced neurite outgrowth; however, microinjection of Ras antibodies into the ts:v-src(3) cells inhibited v-src-induced neurite outgrowth. These studies suggest that there are tyrosine kinases such as Src or Trk which function upstream of Ras. To determine whether there are tyrosine phosphorylation events occurring downstream of Ras, we analyzed NGF-induced tyrosine phosphorylation in a PC12 cell line expressing a dominant interfering variant of Ras (GSrasDN6). Tyrosine phosphorylation of two proteins, related to the MAP kinase family, occurred in a Ras-dependent manner, while activation of Trk or tyrosine phosphorylation of PLC-$\gamma$ was not dependent upon Ras. Thus, there are tyrosine phosphorylation events which occur both upstream and downstream of Ras. (Abstract shortened with permission of author.