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-hybrid 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 ...
compartment of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [(35)S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (~60%) and NOS2 activity (~45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.
Mesh Terms:
Animals, Cell Fractionation, Cell Line, Guanosine Triphosphate, Macrophage Activation, Macrophages, Mice, Nitric Oxide Synthase, Nitric Oxide Synthase Type II, Precipitin Tests, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Recombinant Fusion Proteins, Superoxides, Two-Hybrid System Techniques, rac GTP-Binding Proteins, rac1 GTP-Binding Protein
Am. J. Physiol. Renal Physiol.
Date: Aug. 01, 2001
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