The product of the Raf-1 proto-oncogene is a serine/threonine protein kinase. This kinase can be activated by auto-phosphorylation and phosphorylation by other kinases upon mitogenic stimulation of mammalian cell lines. The phosphorylation events that occur downstream of Raf-1 activation lead to changes in gene expression. Proper structural and functional studies required for understanding the mechanism of activation of Raf-1 kinase awaits an efficient purification scheme. Various sources will be used for such purification, including mammalian cell lines, baculovirus-infected insect cell lines, and Escherichia coli, where Raf-1 protein is overexpressed in appropriate vectors. A preliminary scheme has been developed that yields active Raf-1 kinase. A number of proteins are associated with the Raf-1 kinase upon mitogenic stimulation. Purification and identification of these proteins should provide a clue to mechanisms leading to alteration of gene expression. Several of these proteins have now been partially purified and used for preparation of specific antibodies.
