Tyrosine phosphorylation of the proto-oncoprotein Raf-1 is regulated by Raf-1 itself and the phosphatase Cdc25A.

Phosphorylation and dephosphorylation of proteins provide an ideal regulatory mechanism to elicit major changes in cellular growth and metabolism. The observation that Raf-1, a serine/threonine kinase and the cellular homologue of the mouse sarcoma virus-encoded v-Raf, becomes hyperphosphorylated in response to many signaling events has long suggested that phosphorylation plays a role in regulating Raf-1 activity. The Raf-1 proto-oncoprotein is stimulated by the activation of many tyrosine kinases, including growth factor receptors, members of Janus kinases, and members of the Src kinase family (1, 11, 18, 19, 21, 23–25, 27, 32). Concomitant with activation, Raf-1 is phosphorylated on tyrosines. However, Raf-1 activation under these conditions is rapid and transient in nature, indicating the existence of activating and inactivating mechanisms. The significance of tyrosine phosphorylation of Raf-1 under physiological conditions has been an important issue. Reports in the literature have indicated that stimulation of either platelet-derived growth factor (PDGF), granulocyte-macrophage colony-stimulating factor or interleukin-2 receptors results in activation and tyrosine phosphorylation of Raf-1 in fibroblasts and hematopoietic cells (2, 21, 30). More recently, it was also found that Raf-1 becomes tyrosine phosphorylated and activated in CD4-cross-linked T cells (25), FcRI-cross-linked myeloid cells (23), and squamous carcinoma cells exposed to ionizing radiation (14), suggesting key roles of Raf-1 in activation of hematopoietic cells and in a DNA repair checkpoint. It has also been shown that in transient-expression systems, pp60c-src-mediated activation of Raf-1 involves the phosphorylation of tyrosine residues 340 and/or 341 (17). Mutational and biochemical analyses confirmed that phosphorylation of these two tyrosine residues is necessary to stimulate the catalytic activity of Raf-1 (7). The use of antisera specific for the tyrosine-phosphorylated state of Raf-1 has provided further evidence for the existence of tyrosine phosphorylated Raf-1 (16). Tyrosine kinases such as members of the Src kinase family, Janus kinases, and the PDGF receptor have been implicated in phosphorylating Raf-1 on these tyrosine residues and thereby enhancing its activity (17, 21, 32). However, almost all reports have stated that the level of tyrosine phosphorylation of Raf-1 is low, though higher levels have been seen in rare cases (2, 30). The significance of tyrosine phosphorylation under physiological conditions remains controversial, in part because the levels of tyrosine phosphorylation of Raf-1 are near the limits of detection. The elusive nature of Raf-1 tyrosine phosphorylation in mammalian cells suggests that a rapid and active dephosphorylation process might counterbalance the phosphorylation process. To investigate the mechanisms by which the tyrosine phosphorylation of Raf-1 is regulated, we used both a baculovirus system and PDGF-stimulated mammalian cell lines and found that not only Raf-1 itself but also the phosphatase Cdc25A is involved in this regulation.