Ret-mediated mitogenesis requires Src kinase activity

The proto-oncogene RET encodes a transmembrane growth neurotrophic receptor with tyrosine kinase (TK) activity. RET mutations are associated with seve ral human neoplastic and nonneoplastic diseases, including thyroid papillar y carcinoma, multiple endocrine neoplasia type 2 syndromes, and Hirschsprun g's disease. Activation of receptor TKs results in the binding and activati on of downstream signaling proteins, among which are nonreceptor TKs of the Src family. To test the involvement of c-Src in Ret-mediated signaling, we measured the levels of c-Src activity in NIH3T3 cells coexpressing Ret and the accessory GFR alpha-1 receptor or an epidermal growth factor receptor/ Ret chimeric receptor when the cells were stimulated bg glial cell line-der ived neurotrophic factor or epidermal growth factor, respectively, Ret stim ulation resulted in the activation of c-Src. We also measured the levels of Src kinase activity in cell lines expressing isoforms of the pet receptor activated by different mutations. These cells showed higher Src kinase acti vity than the normal counterpart. Furthermore, we show that Ret is able to associate with the SH2 domain of Src in a phosphotyrosine-dependent fashion . Microinjection of a kinase inactive mutant of c-Src blocked pet-mediated mitogenic effect. These experiments demonstrate that activated Ret is able to bind and stimulate c-Src kinase and that Src activation is essential for the mitogenic activity of Ret.