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 (1007)Y-(1008)Y-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 (1007)Y-(1008)Y-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-(1007)Y-(1008)Y-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 (694)Y-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-(694)Y-STAT5b and phospho-(1007)Y-JAK2 over those arising from GH alone. Nuclear magnetic resonance molecular modeling of natural and 3'-NT- and orthophosphate-substituted peptide analogs of the (1007)Y-(1008)Y 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)Y and/or (1008)Y positions blocked the generation of (1007)Y-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.
Elsasser
Ted H
TH
US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, Maryland 20705, USA. elsasser@anri.barc.usda.gov
Li
Cong-Jun
CJ
Caperna
Thomas J
TJ
Kahl
Stanislaw
S
Schmidt
Walter F
WF
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism* / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D053614:Janus Kinase 2 / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D009566:Nitrates / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D000939:Epitopes; D009566:Nitrates; D011994:Recombinant Proteins; D014443:Tyrosine; D013006:Growth Hormone; D052250:Nitric Oxide Synthase Type III; D053614:Janus Kinase 2; D051057:Proto-Oncogene Proteins c-akt