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.
From: US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, Maryland 20705, USA. elsasser@anri.barc.usda.gov
Endocrinology
- Publish Date: Aug 2007
- ISSN: 0013-7227
- Volume: 148
- Issue: 8
- Pages: 3792-802
- Medium: Print
- Language: English
- Citation (JAMA): Elsasser Ted H, Li Cong-Jun, Caperna Thomas J, et al. 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.. Endocrinology Aug 2007;148:3792-802
Abstract
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.
Mesh Headings (Keywords): Animals, Binding Sites, Cattle, Cells, Cultured, Epitopes, Growth Hormone, Hepatocytes, Janus Kinase 2, Male, Nitrates, Nitric Oxide Synthase Type III, Phosphorylation, Protein Structure, Tertiary, Proto-Oncogene Proteins c-akt, Recombinant Proteins, Signal Transduction, Swine, Tyrosine
Check for Full Text / PubMed Unique Identifier (PMID): 17510232
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