Nerve growth factor activates persistent Rap1 signaling in endosomes

We investigated a role for endogenous Rap1, a small monomeric GTP-binding p rotein of the Ras family, in nerve growth factor (NGF) signaling in PC12 ce lls. Although both epidermal growth factor (EGF) and NGF caused transient a ctivation of Ras, only NGF induced the activation of Rap1. Moreover, Rap1 a ctivation was sustained for hours, an effect that matched the sustained act ivation of the mitogen-activated protein kinase (MAPK) pathway. To investig ate the molecular basis for Rap1 activation, we examined complexes containi ng C3G, a guanine nucleotide exchange factor for Rap1, and CrkL, an adapter protein known to influence Rap1 signaling. NGF induced the formation of a long-lived complex containing C3G/CrkL/Shp2/Gab2/TrkA. Linking the complex to Rap1 activation, we coprecipitated activated TrkA and activated MAPK wit h activated Rap1 in NGF-treated cells. Confocal microscopy and subcellular fractionation showed that activated Rap1 and the other proteins of the sign aling complex were present in endosomes. Pretreatment of PC12 cells with br efeldin A (BFA), which disrupts the Golgi and endosomal compartments, had l ittle effect on Ras activation but strongly inhibited NGF-induced Rap1 acti vation and continuing MAPK activation. We propose that endosomes are a site from which NGF induces the prolonged activation of Rap1 and MAPK.