Growth hormone (GH)-associated nitration of janus kinase-2 at the 1007Y-1008Y epitope impedes phosphorylation at this site:: Mechanism for and impact of a GH, AKT, and nitric oxide synthase axis on GH signal transduction

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 Y-1007-Y-1008-phosphorylation epitope of Janus kinase (JAK)- 2 because of its homology to a defined peptide nitration motif. Using antibodies we developed to the 3' NT-substituted peptide analog of the Y-1007-Y-1008-JAK2 site (nitro-JAK2), we demonstrated a rapid increase in membraneassociated nitro-JAK2 after GH. In vivo ( bovine liver) and in vitro (porcine hepatocytes), GH-induced cellular levels of nitro(1007)Y- Y-1008-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 nitric oxide synthase prior to GH challenge attenuated the increases in nitro-JAK2 predominantly in the membrane subcellular fraction. In instances in which GH effected orthophosphorylation of Y-694- signal transducer and activator of transcription (STAT)- 5b, the addition of AKT and endothelial nitric oxide synthase inhibitors prior to GH significantly increased the levels of phospho-Y-694- STAT5b and phospho-(1007)YJAK2 over those arising from GH alone. Nuclear magnetic resonance molecular modeling of natural and 3'- NT- and orthophosphatesubstituted peptide analogs of the Y-1007-Y-1008 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 (1007)Yand/ or (1008)Ypositions blocked the generation of Y-1007- 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 JAKSTATAKT interactions.