Association of Grb2 with Sos and Ras with Raf-1 upon gamma irradiation of breast cancer cells.

Raf-1 protein serine/threonine kinase has been implicated in growth and damage-responsive signal transduction pathways. Several reports indicate an important role of Ras protein in the growth factor-induced activation of Raf-1. Here we investigated the possible involvement of Ras in ionizing radiation-induced activation of Raf-1. Irradiation of MDA-MB 231 human breast cancer cells caused an increase in GTP-binding and hydrolysis on Ras, and co-immunoprecipitations of endogenous Grb2 with Sos and Raf-1 with Ras. An increase in the level of membrane-bound Raf-1, and tyrosine-phosphorylation of Raf-1 were observed after irradiation. Consistent with these changes, irradiation of cells stimulated the catalytic activity of Raf-1. Finally, radiation treatment of breast cancer cells led to an increase in the phosphorylation and activity of the mitogen-activated protein kinase. Based on these biochemical modifications in vivo, we conclude that Raf-1 functions as an effector of Ras in the radiation-responsive signal transduction pathway leading to the activities of Raf-1 and mitogen-activated protein kinase.