ARS | Publication request: NITRATION OF THE JAK-2 KINASE Y1007-Y1008 PHOSPHORYLATION SITE DURING REPEATED ENDOTOXIN CHALLENGE AND GROWTH HORMONE (GH) ADMINISTRATION

Submitted to: Endocrine Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 2003
Publication Date: June 19, 2003
Citation: Elsasser, T.H., Kahl, S. 2003. Nitration of the JAK-2 kinase Y1007-Y1008 phosphorylation site during repeated endotoxin challenge and growth hormone (GH) administration [abstract]. Endocrine Society Annual Meeting. p. 149.

Technical Abstract: Oxynitrogen intermediates such as peroxynitrite (ONOO-), formed through the reaction of nitric oxide with superoxide anion, quickly nitrate the phenolic ring of tyrosine residues in proteins. Past work in our laboratory identified significant levels of hepatic protein nitration that develop over time after the administration of low levels (2.5 mg/kg) E. coli endotoxin. Further to this point, administration of vitamin E, predominantly a membrane-based antioxidant, reduced the magnitude of this nitration, suggesting that much of the nitration reaction may have occurred in or in close proximity to the plasma membrane. The double endotoxin challenge (2.5 mg/kg, i.v. 4 days apart) in unrestrained year-old female calves was used to determine if the Y1007-Y1008 phosphorylation activation site of JAK-2 kinase was an in vivo nitration target during response to endotoxin in light of previous observations of JAK-2 nitration being detected by generalized antinitrotyrosine western blot of anti-JAK-2-immunoprecipitated protein. Nitration of JAK-2 was assessed in hepatic tissue harvested under local anesthetic approximately 6 hours after the second day s administration of endotoxin. In addition, JAK-2 nitration was also assessed in a separate group of similarly reared and handled animals treated for 12 days with recombinant bovine GH (100 mg/kg, i.m., n=6) or saline (n=6) but no endotoxin, with biopsy samples similarly harvested and processed. Y1007-Y1008-nitrated JAK-2 was detected by western blot of protein homogenates and immunohistochemistry performed on Bouin s fixed biopsy core specimens using a highly specific rabbit antibody directed to a synthetic nitration-modified JAK-2 kinase peptide region: LPQDE(nitroY1007)(nitroY1008)KVKEPGESPIFW. Nitrated JAK-2 was detected in endotoxin-challenged animals and control animals receiving the 12-day GH injections. Interestingly, the Y1007-Y1008 nitrated JAK-2 detected in the12-day GH-injected animals corresponded with measured increases (over nonGH injected controls) in basal level of constitutive endothelial Type-3 nitric oxide synthase (caveolae-associated) and hepatic protein nitration. The data suggest that tyrosine nitration of JAK-2 kinase in the Y1007-Y1008 phosphorylation site may participate in compromised GH signal transduction and disruption of metabolic control by GH during oxidative disease stress. In addition, nitration of JAK-2 may be a regulatory phosphorylation modulator under normal circumstances.