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Title: Nitration of JAK-2 at the 1007Y-1008Y activation epitope impedes phosphorylation at this site: defining a GH, AKT/protein kinase B and nitric oxide synthase axis

Author
item Elsasser, Theodore
item Li, Congjun - Cj
item Caperna, Thomas
item Kahl, Stanislaw
item Schmidt, Walter

Submitted to: Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2007
Publication Date: 5/18/2007
Citation: Elsasser, T.H., Li, C., Caperna, T.J., Kahl, S., Schmidt, W.F. 2007. GH-associated nitration of JAK2 at the 1007Y-1008Y epitope impedes phosphorylation at this site: Mechanism for and impact of a GH, AKT and nitric oxide synthase (NOS) axis on GH signal transduction. Endocrinology. 148:3792-3802. Available: http://endo.endojournals.org.

Interpretive Summary: Hormones exert their effects on cell metabolism by interacting with specific receptors for that hormone. The effects within the cell are transmitted to the nucleus by a process called signal transduction where several different proteins are modified by the attachment of a phosphate group to activate an enzymatic property of the protein. We have shown for the first time that the generation of a nitrate in the place where the phosphate was to have gone, blocks the function of the nitrated protein. We further have shown that this is a phenomenon initiated by growth hormone and may participate in some of the mechanisms that limit the extent of the actions of growth hormone. Our data have shown that a feature critical to the generation of this nitration by growth hormone is that the reactants need to be closely positioned next to each other and we have demonstrated that all of the necessary reactants can be assembled in membrane structures called caveolae. The observations have provided new insights to how hormone activity is regulated and may open new visions as regards how disease mechanisms work.

Technical Abstract: Generalized liver protein tyrosine nitration (3’-nitrotyrosine, 3’-NT) increases in vivo after GH injection with immunohistocellular patterns strikingly similar to those we observed for a specific nitration of JAK2 at its 1007Y-1008Y regulatory phosphorylation epitope following proinflammatory challenge. Here we investigated whether GH directs the generation of 3’-NT at the 1007Y-1008Y site and questioned how such a specific nitration might affect the phosphorylation capacity of tyrosines at this regulatory site. Using antibodies we developed to the 3’NT-substituted peptide analog of the 1007Y-1008Y-JAK-2 site, we demonstrated a rapid increase in membrane-associated 1007Y-1008Y-JAK-2 nitration after GH. Peak levels of phosphoJAK2 occurred sooner after GH than those of nitrated JAK2 in bovine liver in vivo and in vitro (porcine hepatocytes); nitrated JAK2 persisted for a significantly longer time than did phosphoJAK2. Treatment of cultured cells with selective inhibitors of AKT or endothelial nitric oxide synthase (eNOS) prior to GH challenge attenuated or blocked the increases in 3’-NT in JAK-2 predominantly in membrane subcellular compartment fractions. Whereas GH effected orthophosphorylation of 694Y -STAT5b, when AKT or eNOS inhibitors were added prior to GH, the levels of phospho-694Y-STAT5b and phospho-1007Y-JAK-2 were significantly increased over those arising from GH alone. NMR-molecular modeling analysis of natural and 3’-NT- and orthophosphate-substituted 20 amino acid analogs of the 1007Y-1008Y-JAK-2 phosphorylation 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 1007Y and/or 1008Y positions blocked the generation of 1007Y-phosphotyrosine.