Recent studies indicate that Rac1-regulated generation of reactive oxygen species (ROS) may be critical to mitogenic signaling. The generation of ROS downstream of the GTP-binding protein Rac1 is known to be crucial to the microbicidal activity of phagocytes. Little is known about the signaling role ROS play in other cell types and the protein targets of ROS are not known. Protein tyrosine phosphatases are good candidates, since oxidation of the catalytic cysteine can reversibly inhibit phosphatases. This mechanism may also contribute to the progression of acute myelogenous leukemia which is characterized by the expression of bcr-abl and alterations in the activity of a protein tyrosine phosphatase. An inhibition of protein tyrosine phosphatase activity could contribute to positive signals in growth factor and bcr-abl signaling. I propose to screen for bcr-abl and growth factor-induced phosphatase inactivation, as a potential signaling pathway mediated by ROS, using a modified in-gel phosphatase assay which detects inhibited protein tyrosine phosphatases. Upon detection and identification of affected phosphatases, I will investigate the signaling events that lie upstream of ROS production by determining which guanine nucleotide exchange factors that regulate Rac1 activation lead to ROS production. Downstream substrates of ROS-inactivated phosphatases will be identified using substrate trapping mutants of the phosphatases. Overall these studies are designed to identify and characterize a ROS-regulated phosphatase activity that regulates the phosphorylation of proteins involved in the control of cell growth.
