A generalized increase in liver protein tyrosine nitration (3'-nitrotyrosine, 3'-NT) occurs after GH injection in a time frame consistent with observed acute GH hyporesponsiveness. Here we investigated whether the GH-associated nitration process might be targeted to the ₁₀₀₇Y-₁₀₀₈Y-phosphorylation epitope of JAK2 because of its homology to a defined peptide nitration motif. Using antibodies we developed to the 3'NT-substituted peptide analog of the ₁₀₀₇Y-₁₀₀₈Y-JAK2 site (nitro-JAK2), we demonstrated a rapid increase in membrane-associated nitro-JAK2 after GH. In vivo (bovine liver) and in vitro (porcine hepatocytes) GH-induced cellular levels of nitro-₁₀₀₇Y-₁₀₀₈Y-JAK2 persisted significantly longer after a stimulatory GH pulse than did levels of phospho-JAK2. Treatment of cultured cells with inhibitors of AKT or endothelial NOS (eNOS) prior to GH challenge attenuated the increases in nitro-JAK2 predominantly in the membrane subcellular fraction. Where GH effected orthophosphorylation of ₆₉₄Y -STAT5b, the addition of AKT and eNOS inhibitors prior to GH, significantly increased the levels of phospho-₆₉₄Y-STAT5b and phospho-₁₀₀₇Y-JAK2 over those arising from GH alone. NMR-molecular modeling of natural and 3'-NT- and orthophosphate-substituted peptide analogs of the ₁₀₀₇Y-₁₀₀₈Y site demonstrated significant effects of 3'-nitration on the planar orientation and intramolecular stabilizing points of the affected tyrosines. When these peptides were used as substrates for in vitro tyrosine kinase phosphorylation reactions, 3'-NT in the ₁₀₀₇Y and/or ₁₀₀₈Y positions blocked the generation of ₁₀₀₇Y-phosphotyrosine. The data suggest that the nitration of JAK2 may act as an inhibitory counterpart to phosphorylation activation reflecting a very localized break on the progression of GH signal transduction processes spanning JAK-STAT-AKT interactions.