Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages

Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hy brid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264 .7 macrophages. The two proteins colocalized in an intracellular compartmen t of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygena se domain was necessary and sufficient for the interaction. [S-35] methioni ne-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, ca lmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (similar to 60%) and NOS2 activity (similar to 45%) without measurably affecting NOS2 protei n abundance and led to a redistribution of NOS2 to a high-speed Triton X-10 0-insoluble fraction. We conclude that Rac1 and Rac2 physically interact wi th NOS2 in activated macrophages and that the interaction with Rac2 correla tes with a posttranslational stimulation of NOS2 activity and likely its sp atial redistribution within the cell.