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

钙激活激素和胰升糖素对肝脏Na+/K+-ATPase有激活作用。在……里面 大鼠肝脏，这种刺激发生得相当快(即不到30秒) 而不是次要于钠内流的增加。来自几个人的证据 包括我们自己的实验室已经发现了甘油二酯/蛋白质 激酶C途径(钙激活激素)与cAMP/蛋白激酶A 在这些药物刺激Na+/K+-ATPase的过程中，可能存在一种新的途径(高血糖素)。 鉴于蛋白质磷酸化在细胞周期中的潜在调节作用 激素依赖的Na+/K+-ATPase激活，我们建议测试 Na+/K+-ATPase被磷酸化和功能调控的假说 通过两种激酶对这种酶的调节，将有两个特定的目的 承担：第一个目标将是描述 特殊改变蛋白激酶C和cAMP依赖的干预措施 蛋白激酶活性或抑制蛋白磷酸酶，对活性的影响 Na+/K+-ATPase及其磷酸化程度 α亚基和β亚基。这将包括药物操纵 Na+/K+-ATPase活性及其与特异性激活剂的磷酸化 CAMP依赖的蛋白激酶和蛋白激酶C 这两种酶的抑制剂阻断激素依赖的激活 此外，还将研究Na+/K+-ATPase的磷酸化。我们会走得更远 试图确定蛋白质磷酸酶抑制剂，己二酸， 会延长或模仿对激素的活性和磷酸化反应。 新鲜分离的大鼠肝细胞及肝细胞培养 在蛋白激酶C活性下调的情况下， 实验。在所有拟议的研究中，时间和注意力 为了将这两个响应关联起来，将获得相关的更改。 将测量哇巴因敏感的86Rb+摄取以监测Na+/K+-ATPase 酶活性和Na+/K+-ATPase亚单位磷酸化 通过监测32P在α和β中的掺入情况进行评估 亚基(即亚基将从洗涤剂中免疫沉淀 肝细胞[~(32)P]放射平衡后的提取液 聚丙烯酰胺凝胶电泳法和放射自显影技术)。初步 研究已经建立了一种二维凝胶方法，将 Na+/K+-ATPase亚基和识别亚基的抗血清。这个 这些研究的第二个目标是确定任何氨基酸残基 Na+/K+-ATPase被这些酶磷酸化，并确定它们的 位于α-和β-亚基的一级结构中。为 为此，免疫沉淀的[32P]-磷酸蛋白对应于 α亚基和β亚基将被部分水解，并受到 磷酸氨基酸分析。I随后的研究放射性标记的亚基 将在电泳法之前进行蛋白水解酶消化， Western blotting和Na+/K+-ATPase亚基序列分析。《长河》 本项目的学期目标是阐明分子机制。 负责激素对Na+/K+-ATPase的刺激，以便 确定它们在调节肝脏代谢中的作用。

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.