Submitted to: Endocrine Society Meeting
Publication Type: Abstract only
Publication Acceptance Date: 3/15/2006
Publication Date: 5/1/2006
Citation: Elsasser, T.H., Caperna, R.J., Li, C., Kahl, S. 2006. Growth hormone (GH) directs nitration of the tyrosine phosphorylation site of JAK-2 (1007-1008Y) in hepatocytes through an AKT/Phosphorylase Kinase B activation of endothelial nitric oxide synthase (eNOS) [abstract]. Proceedings of the Annual Meeting of the Endocrine Society. p. 44. Interpretive Summary:
Technical Abstract: The phenolic ring of tyrosine (Y) has been identified as a major target for attack by the reactive nitrogen anion peroxynitrite (ONOO-), a product of the interaction of nitric oxide (NO) with superoxide anion. ONOO- places the nitrate moiety at the 3'-position on the Y-ring preventing the phosphorylation of the ring at the ortho-position. Typically, protein Y-nitration is associated with tissue pathology. However, recent data suggest that some levels of Y-nitration may develop under normal health circumstances and play a role in cytokine signal transduction. Previously, we identified such a nitration at the 1007-1008Y activation epitope of JAK-2 following endotoxin administration in vivo with associated decreases in the ability for GH to regulate IGF-1. Significant to that observation was the fact that the nitrated JAK-2 colocalized in membrane caveolae along with eNOS. Our aim presently was to identify critical control points that mediate a GH-induced Y-nitration of JAK-2. Porcine hepatocytes, isolated by collagenase processing, were seeded into flasks (for Western blot) or chambered microscope slides (for quantitative immunohistochemistry, IHC). Cells were challenged with 100 ng/ml recombinant porcine GH and harvested or fixed immediately or at 10 and 30 min following GH. Additional cell cultures were treated with 1mM NG-monomethyl-L-arginine (eNOSI, eNOS inhibitor) or 10 'M AKT inhibitor (AKTI, Calbiochem 124005) for 30 min prior to GH. IHC image analysis of hepatocytes indicated that GH induced a burst of JAK-2 phosphorylation by 10 min that resolved to baseline by 30 min. Using an affinity purified antibody to a nitrated analog of the 1007-1008Y-JAK-2 activation epitope (1001LPQDKE-nitro1007Y-nitro1008-Y-KVKEPGESPIFW1020), we demonstrated that the nitration at this site continued to increase 30 min after GH. AKTI decreased eNOS phosphorylation after GH by 90%; JAK-2 nitration was decreased 70% with AKTI and totally blocked in the presence of eNOSI. The data are consistent with the concept that the ability for GH to nitrate JAK-2 at the 1007-1008Y phosphorylation site is driven, at least in part, through GH phosphorylation activation of eNOS via an effect on AKT and that the presence of the nitration persists beyond a phosphorylation. These data complement and extend prior in vivo data from our lab suggesting that epitope-specific JAK-2 nitrations may complement phosphorylation in a very localized fine tuning of signal transduction processes.