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

本项目的总体目标是验证磷酸化蛋白接头14 - 3 - 3.a在广盐性硬骨鱼盐度驯化过程中控制鳃上皮重组的假设。 具体而言，本项目将调查渗透调节14 - 3 - 3.a在多个水平（表达，翻译后调节，和compartmentalization）和它的相互作用与丝裂原活化蛋白激酶（MAPK）在适应广盐鱼类底鳃上皮细胞的机制heteroclitus。 本项目的重点是详细研究14 - 3 - 3.a的渗透调节。这是一个关键问题，因为14-3-3蛋白对于基于磷酸化的信号转导途径具有普遍和特别的重要性。这些蛋白质是一种新型的分子衔接子，其调节参与环境调节的信号传导途径、细胞分化和细胞周期控制的几乎所有重要组分之间的相互作用（Fu等人，2000年）。14-3-3蛋白质通过结合和螯合在丝氨酸或苏氨酸上磷酸化的蛋白质来调节细胞活性和功能。这些分子可能控制着广盐性硬骨鱼在盐度适应过程中鳃上皮的重组，因为1）蛋白磷酸化是盐度感觉信号转导的主要机制，2）14-3-3蛋白参与了许多促有丝分裂、离子转运和细胞分化途径的调节。 大量的初步数据为拟议项目提供了坚实的基础，并为深入实现该项目的所有目标提供了全面的基础。广盐性鱼类F. heteroclitus（GB AF 302039）含有一个开放阅读框，编码第一个已知的来自鱼类的14-3-3蛋白，命名为14 - 3 - 3. a. 最近的数据提供了明确的证据表明，14 - 3 - 3.a基因强烈诱导的鳃上皮细胞的F。从海水（SW）向淡水（FW）转移。 MAPK信号转导通路ERK、JNK和p38在草鱼鳃上皮细胞中的丰度和活性。从SW转移到FW，反之亦然的heteroclitus表明，所有MAPK的活性强烈调制广盐性鱼类的盐度驯化过程中。 目的是详细分析14 - 3 - 3.a对广盐性鱼类鳃上皮细胞的渗透调节作用。1）了解14 - 3 - 3.a mRNA和蛋白表达的渗透调节模式。2)研究渗透压摩尔浓度变化如何影响14 - 3 - 3.a的翻译后修饰。3)了解14 - 3 - 3.a在细胞和亚细胞中的定位及其在体内的调节作用。4)确定14 - 3 - 3.a是否与适应性鳃上皮中的MAPK相互作用。 解决这些目标的方法将基于重组DNA方法学;蛋白质印迹法、北方印迹法、二维电泳，然后是MALDI-TOF质谱法、免疫细胞化学、免疫沉淀、下拉测定和激酶测定。 预计该项目将显著推进细胞神经感觉信号转导的知识。 这将为研究鱼类的感觉信号转导提供一种新的广盐性动物模型。广盐性鱼类有一个肾外运输上皮，即直接暴露于外部环境的鳃上皮，可以在完整的动物体内进行研究。与哺乳动物肾细胞不同，鱼鳃上皮细胞周围介质的渗透压可以在体内准确和即时地操纵。 这项研究应提供全面的渗透调节的14 - 3 - 3.a，一个很好的候选分子，用于管理的多方面的神经感觉信号转导和细胞分化，在适应鳃上皮细胞的广盐性硬骨鱼暴露于盐度变化。

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.