Elsasser Ted H, Li Cong-Jun, Caperna Thomas J, Kahl Stanislaw, Schmidt Walter F
US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, Maryland 20705, USA.
Endocrinology. 2007 Aug;148(8):3792-802. doi: 10.1210/en.2006-1736. Epub 2007 May 17.
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.
生长激素（GH）注射后，肝脏蛋白酪氨酸硝化（3'-硝基酪氨酸，3'-NT）普遍增加，其时间框架与观察到的急性GH低反应性一致。在此，我们研究了由于GH相关的硝化过程与特定肽硝化基序具有同源性，它是否可能靶向Janus激酶（JAK）-2的（1007）Y-（1008）Y-磷酸化表位。使用我们开发的针对（1007）Y-（1008）Y-JAK2位点的3'NT取代肽类似物（硝基-JAK2）的抗体，我们证明了GH后膜相关硝基-JAK2迅速增加。在体内（牛肝脏）和体外（猪肝细胞），刺激GH脉冲后，GH诱导的硝基-（1007）Y-（1008）Y-JAK2细胞水平持续时间明显长于磷酸化-JAK2水平。在GH刺激前用AKT或内皮型一氧化氮合酶抑制剂处理培养细胞，主要减弱了膜亚细胞组分中硝基-JAK2的增加。在GH使信号转导子和转录激活子（STAT）-5b的（694）Y发生正磷酸化的情况下，在GH之前添加AKT和内皮型一氧化氮合酶抑制剂，与单独使用GH相比，显著增加了磷酸化-（694）Y-STAT5b和磷酸化-（1007）Y-JAK2的水平。对（1007）Y-（1008）Y位点的天然、3'-NT和正磷酸盐取代肽类似物进行核磁共振分子建模，结果表明3'-硝化对受影响酪氨酸的平面取向和分子内稳定点有显著影响。当这些肽用作体外酪氨酸激酶磷酸化反应的底物时，（1007）Y和/或（1008）Y位置的3'-NT阻断了（1007）Y-磷酸酪氨酸的生成。数据表明，JAK2的硝化可能作为磷酸化激活的抑制对应物，反映了跨越JAK-STAT-AKT相互作用的GH信号转导过程进展中的一个非常局部的中断。
