REGULATION OF HEPATIC NA+/K+ -ATPASE BY PROTEIN KINASES

钙动员激素和胰高血糖素刺激肝Na +/K +-ATP酶。 在 大鼠肝脏，这种刺激发生得相当快（即小于30秒） 并且不继发于增加的Na-流入。 几个证据 包括我们自己在内的实验室已经暗示了甘油二酯/蛋白质 激酶C途径（Ca2+动员激素）和cAMP/蛋白激酶A 途径（胰高血糖素）在这些药物刺激Na +/K +-ATP酶中的作用。 鉴于蛋白质磷酸化在细胞内的潜在调节作用， Na +/K +-ATP酶的激素依赖性激活，我们建议测试 Na~+/K~+-ATP酶磷酸化和功能调节假说 通过两种激酶调节这种酶， 承诺：第一个目标将是描述的影响， 干预，这具体改变蛋白激酶C和cAMP依赖 蛋白激酶活性或抑制蛋白磷酸酶活性， Na~+/K~+-ATP酶及其磷酸化程度 α和β亚基。 这将包括药理学操作， Na~+/K~+-ATP酶活性和磷酸化与特异性激活剂的关系 cAMP依赖性蛋白激酶和蛋白激酶C。 具体能力 这两种激酶的抑制剂，以中断激素依赖性激活 Na~+/K~+-ATP酶的磷酸化也将被研究。 此外，我们将 试图确定蛋白磷酸酶抑制剂，冈田酸， 将延长或模拟对激素的活性和磷酸化反应。 新鲜分离的大鼠肝细胞以及肝细胞的细胞培养物 蛋白激酶C活性下调的细胞将用于这些研究。 实验 在所有拟议的研究中， 将获得相关的变化，以便使两个响应相关。 将测量哇巴因敏感的86 Rb+摄取以监测Na +/K +-ATP酶 酶活性和Na +/K +-ATP酶亚基磷酸化将是 通过监测32P掺入α和β 亚基（即亚基将从洗涤剂中免疫沉淀 肝细胞提取物与[32P]放射平衡的ATP池， 聚丙烯酰胺凝胶电泳和放射自显影）。 初步 研究已经建立了二维凝胶方法 Na +/K +-ATP酶亚基和识别该亚基的抗血清。 的 这些研究的第二个目的是鉴定 Na +/K +-ATP酶的磷酸化，并确定其 在α和β亚基的一级结构中的位置。 为 为此，免疫沉淀的[32P]-磷蛋白对应于 α和β亚基将被部分水解， 磷酸氨基酸分析 I随后的研究放射性标记亚基 将在电泳之前进行蛋白水解消化， Na~+/K~+-ATP酶亚基的Western印迹和序列分析 长 本项目的长期目标是阐明 负责通过激素刺激Na +/K +-ATP酶， 确定其在肝脏代谢调节中的作用。

Ca2+-mobilizing hormones and glucagon stimulate hepatic Na+/K+-ATPase. In rat liver, this stimulation occurs fairly rapidly (i.e. less than 30 sec) and is not secondary to increased Na-influx. Evidence from several laboratories including our own has implicated the diacylglycerol/protein kinase C pathway (Ca2+-mobilizing hormones) and cAMP/protein kinase A pathway (glucagon) in the stimulation of the Na+/K+-ATPase by these agents. In view of the potential regulatory role of protein phosphorylation in hormone dependent activation of Na+/K+-ATPase, we propose to test hypothesis that Na+/K+-ATPase is phosphorylated and functionally regulated by two kinases in the regulation of this enzyme two specific aims will be undertaken: The first aim will be to characterize the effect of interventions, which specifically alter protein kinase C and cAMP-dependent protein kinase activity or inhibit protein phosphatases, on the activity of Na+/K+-ATPase as well as the extent of phosphorylation of Na+/K+-ATPase alpha and beta subunits. This will include pharmacologic manipulation of Na+/K+-ATPase activity and phosphorylation with specific activators of cAMP-dependent protein kinase and protein kinase C. The ability specific inhibitors of these two kinases to interrupt hormone dependent activation and phosphorylation of Na+/K+-ATPase will also be studied. Further we will seek to determine whether the protein phosphatase inhibitor, okadiac acid, will prolong or mimic activity and phosphorylation responses to hormones. Freshly isolated rat hepatocytes as well as cell cultures of hepatocytes with down-regulated protein kinase C activities will be utilized for these experiments. In all of the proposed studies, time and concentration dependent changes will be obtained in order to correlate the two responses. Ouabain-sensitive 86Rb+-uptake will be measured to monitor Na+/K+-ATPase enzymatic activity and Na+/K+-ATPase subunit phosphorylation will be evaluated by monitoring the incorporation of 32P into the alpha and beta subunits (i.e. the subunits will be immunoprecipitated from detergent extracts of hepatocytes with [32P] radioequilibrated ATP pools prior to polyacrylamide gel electrophoresis and autoradiography). Preliminary studies have established a two dimensional gel methodology which separates Na+/K+-ATPase subunits and antisera which recognize the subunits. The second aim of these studies is to identify any amino acid residues in Na+/K+-ATPase which are phosphorylated by these kinases and ascertain their location within the primary structure of the alpha- and beta-subunits. For this purpose, immunoprecipitated [32P]-phosphoproteins corresponding to the alpha and beta subunits will be partially hydrolyzed and subjected to phosphoamino acid analysis. I subsequent studies radiolabelled subunits will be subjected to proteolytic digestion prior to electrophoresis, Western blotting and sequence analysis of Na+/K+-ATPase subunits. The long term goals of this project are to elucidate the molecular mechanisms responsible for stimulation of Na+/K+-ATPase by hormones in order to determine their role in the regulation of liver metabolism.