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.
Kuncewicz
T
T
Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.
Balakrishnan
P
P
Snuggs
M B
MB
Kone
B C
BC
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
D006160:Guanosine Triphosphate; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C496317:Nos2 protein, mouse