Submitted to: Endocrine Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2004
Publication Date: 4/1/2004
Citation: Elsasser, T.H., Kahl, S., Li, C., Wilkins, B. 2004. Growth hormone administration promotes a temporally-defined low-level nitration of the Y1007-Y1008 activation site of JAK-2 kinase in vivo and invitro [abstract]. Endocrine Society Program and Abstracts. p. 245.
Technical Abstract: Recently, we identified a membrane caveolar-situated, specific nitration (NO3Y1007- NO3Y1008 = nitro-JAK-2) of the Y1007-Y1008 phosphorylation activation site of JAK-2 kinase in calf liver during the oxidative stress of endotoxin challenges and concomitant generation of the nitrating anion peroxynitrite (ONOO-). We further observed a slight but repeatably detectable increase in the level of nitro-JAK-2 in liver homogenates from calves that chronically had been treated with growth hormone (GH) compared to nitro-JAK-2 levels of saline-treated calves. The objective of the present study was to determine whether a GH challenge alone would effect acutely a nitration of JAK-2 at this Y1007-Y1007 site. Liver tissue was obtained from calves via transcutaneous biopsy. Tissue sample cores were collected serially from the same animal (n=3) at time 0 and at 2, 5, 15, 30 and 60 min following an iv. injection of recombinant bovine GH (100 micrograms/kg wt). Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Changes in levels of nitro-JAK-2 were estimated by quantitative immunohistochemistry using an antibody generated to a 3-nitrotyrosine-analog of the natural JAK-2 activation sequence (LPQDE NO3Y1007-NO3Y1008 KVKEPGESPIFW). Antigen localization, detected using biotinylated tyramide-enhancement immunostaining, was digitally photographed; staining intensity was estimated using a color-specific (diaminobenzidine brown) pixel-quantifying image analysis. Regional increases in nitro-JAK-2 were detectable at 15 min whereas increases in phosphorylated-JAK-2, used as a positive control for response to GH, were evident as early as 5 min. The GH-related increases in JAK-2 nitration (approximately 2-fold) were predominantly, though not exclusively, localized to the cellular regions surrounding the central veins. Testing this response further, we cultured MDBK epithelial cells, challenged them with 500 ng GH/ml media, and harvested cells at 0, 5, 10, 15, 30, and 60 min relative to challenge application. PAGE separations of sonicated MDBK cell homogenates were positive at the 10 min point for a nitro-JAK-2 (kD=129) by western blot using the anti-nitro-JAK-2 antibody. The data indicate that GH administration is associated with a temporally and spatially defined low-level increase in a nitrated Y1007-Y1008 epitope of JAK-2. This data suggests a potential unique role for nitration reactions in modulation of GH signal transduction pathways.