Role of Protein Phosphorylation for Osmotic Stress Adaptation of a Euryhaline Teleost

该项目的总体目标是检验这一假说，即磷酸蛋白适配器14-3-3.a在真盐硬骨鱼的盐驯化过程中控制着鳃上皮的重组。具体地说，本项目将在多个水平上研究14-3-3.a的渗透调节机制(表达、翻译后调节和区域化)，以及它与异地锦鱼适应的鳃上皮中丝裂原激活蛋白激酶(MAPKs)的相互作用。本项目的重点是对14-3-3.a的渗透调节进行详细的研究。这是一个关键问题，因为14-3-3蛋白对于以磷酸化为基础的信号转导途径具有普遍而特殊的重要性。这些蛋白质是一种新型的分子适配器，可以调节几乎所有参与环境调节的信号通路、细胞分化和细胞周期控制的重要组成部分之间的相互作用(Fu等人，2000)。14-3-3蛋白通过结合和隔离丝氨酸或苏氨酸上的磷酸化蛋白来调节细胞的活动和功能。这些分子可能在真盐硬骨鱼的盐度适应过程中控制着鳃上皮的重组，因为1)蛋白质磷酸化是渗透感觉信号转导的主要机制，2)14-3-3蛋白参与了许多有丝分裂、离子运输和细胞分化途径的调节。大量的初步数据为拟议的项目提供了坚实的基础，并为深入实现该项目的所有目标奠定了全面的基础。克隆并测序了一条新的真盐目鱼类F.astoclitus(GB AF302039)的新基因，它含有一个开放阅读框，编码从鱼类中首次发现的14-3-3蛋白，命名为14-3-3.a。最近的研究表明，14-3-3.a基因在从海水(Sw)转移到淡水(FW)的异地对虾(FW)的鳃上皮中被强烈诱导。Sw转FW和FW转MAPKs ERK、JNK和p38的丰度和活性表明，在盐度驯化过程中，所有MAPKs的活性都受到强烈的调节。本研究旨在对14-3-3.a在真盐目鱼类鳃上皮细胞中的渗透调节进行详细的分析：1)了解14-3-3.a的mRNA和蛋白表达的渗透调节规律；2)研究渗透压变化对14-3-3.a翻译后修饰的影响。3)了解14-3-3.a的细胞和亚细胞定位及其渗透调节机制。4)确定14-3-3.a是否与渗透适应的鳃上皮中的MAPKs相互作用。解决这些目标的方法将基于重组DNA方法学、Western blotting、Northern blotting、双向电泳和MALDI-TOF质谱学、免疫细胞化学、免疫沉淀、下拉试验和激酶分析。预计该项目将极大地促进对细胞渗透感觉信号转导的了解。它将引入一种新的泛盐目鱼类模型来研究渗透感觉信号转导。真盐碱鱼有一种肾外运输上皮，即直接暴露在外部环境中的鳃上皮，可以在完整的动物体内进行研究。与哺乳动物肾脏细胞不同，鱼鳃上皮细胞周围的培养液的渗透压可以在体内准确和瞬时地操纵。14-3-3.a是调节渗透感觉信号转导和细胞分化的一个很好的候选分子，在适应盐度变化的真盐硬骨鱼的鳃上皮细胞的过程中，14-3-3.a是一个很好的候选分子，这项研究将为我们提供全面的了解。

The overall objective of this project is to test the hypothesis that the phospho-protein adaptor 14-3-3.a controls the reorganization of the gill epithelium during salinity acclimation of euryhaline teleosts. Specifically, this project will investigate the mechanisms of osmotic regulation of 14-3-3.a at multiple levels (expression, posttranslational regulation, and compartmentalization) and its interaction with mitogen-activated protein kinases (MAPKs) in the adapting gill epithelium of the euryhaline fish Fundulus heteroclitus. The focus of this project is a detailed investigation of the osmotic regulation of 14-3-3.a. This is a critical issue because 14-3-3 proteins are of general and extraordinary importance for signal transduction pathways that are based on phosphorylation. These proteins are a novel type of molecular adaptor that modulates interactions among virtually all important components involved in environmentally regulated signaling pathways, cell differentiation, and cell cycle control (Fu et al., 2000). 14-3-3 proteins regulate cellular activity and function by binding and sequestering proteins phosphorylated on serine or threonine. These molecules likely control the reorganization of gill epithelium during salinity adaptation of euryhaline teleosts because 1) protein phosphorylation is a major mechanism of osmosensory signal transduction and 2) 14-3-3 proteins are involved in the regulation of many mitogenic, ion transport, and cell differentiation pathways. Substantial preliminary data provide a strong foundation for the proposed project and represent a comprehensive basis for addressing all aims of this project in depth. A cloned and sequenced novel cDNA from the euryhaline fish F. heteroclitus (GB AF302039) contains an open reading frame encoding the first known 14-3-3 protein from fish that is named 14-3-3.a. Recent data provide clear evidence that the 14-3-3.a gene is strongly induced in gill epithelium of F. heteroclitus transferred from seawater (SW) to fresh water (FW). The abundance and activities of the MAPKs ERK, JNK, and p38 in gill epithelium of F. heteroclitus transferred from SW to FW and vice versa show that the activity of all MAPKs is strongly modulated during salinity acclimation of euryhaline fish. The aims focus on a detailed analysis of the osmotic regulation of 14-3-3.a in gill epithelium of the euryhaline fish F. heteroclitus: 1) To know the profile of osmotic regulation of 14-3-3.a mRNA and protein expression. 2) To investigate how osmolality changes affect posttranslational modification of 14-3-3.a. 3) To know the cellular and subcellular localization of 14-3-3.a and how it is osmotically regulated. 4) To determine whether 14-3-3.a interacts with MAPKs in the osmotically adapting gill epithelium. The approach for addressing these aims will be based on recombinant DNA methodology; western blotting, northern blotting, two-dimensional electrophoresis followed by MALDI-TOF mass spectrometry, immunocytochemistry, immunoprecipitation, pull-down assays, and kinase assays. It is anticipated that this project will significantly advance the knowledge of cellular osmosensory signal transduction. It will introduce a novel euryhaline fish model to osmosensory signal transduction research. Euryhaline fishes have an extrarenal transport epithelium the gill epithelium that is directly exposed to the external milieu and can be studied in intact animals in vivo. Unlike for mammalian renal cells, the osmolality of the medium surrounding fish gill epithelial cells can be accurately and instantaneously manipulated in vivo. This research should provide comprehensive insight into the osmotic regulation of 14-3-3.a, an excellent candidate molecule for governing many aspects of osmosensory signal transduction and cell differentiation in adapting gill epithelial cells of euryhaline teleosts exposed to salinity changes.