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

本课题的总体目标是验证磷酸蛋白适配器14-3-3的假设。在盐驯化过程中，A控制鳃上皮的重组。具体而言，本项目将研究14-3-3的渗透调节机制。a在多水平（表达、翻译后调控和区隔化）及其与丝裂原活化蛋白激酶（MAPKs）在广盐鱼底异羞耻鱼适应性鳃上皮中的相互作用。本课题的重点是对14-3-3.a的渗透调控进行详细的研究。这是一个关键问题，因为14-3-3蛋白在基于磷酸化的信号转导途径中具有普遍和非凡的重要性。这些蛋白质是一种新型的分子接头，可以调节环境调节信号通路、细胞分化和细胞周期控制中几乎所有重要成分之间的相互作用（Fu et al., 2000）。14-3-3蛋白质通过结合和隔离丝氨酸或苏氨酸磷酸化的蛋白质来调节细胞的活性和功能。这些分子很可能在盐盐硬骨鱼适应过程中控制鳃上皮的重组，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蛋白参与许多有丝分裂、离子转运和细胞分化途径的调节。大量的初步数据为拟议的项目提供了坚实的基础，并为深入解决该项目的所有目标提供了全面的基础。经克隆和测序的一份来自欧盐鱼F. heteroclitus （GB AF302039）的新cDNA包含一个开放阅读框，编码已知的第一个来自鱼类的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，证明14-3-3。一个基因在海水（SW）转移到淡水（FW）的异交梭菌（F. heteroclitus）鳃上皮中被强烈诱导。从SW转移到FW和反之转移的F. heteroclitus鳃上皮中mapk ERK， JNK和p38的丰度和活性表明，所有mapk的活性在盐盐鱼的盐度驯化过程中受到强烈调节。重点对14-3-3的渗透调控进行了详细分析。1)了解14-3-3渗透调控的概况。a mRNA和蛋白的表达。2)探讨渗透压变化对14-3-3.a翻译后修饰的影响。3)了解14-3-3的细胞和亚细胞定位。以及它是如何被渗透调节的。4)确定是否14-3-3。a在渗透适应性鳃上皮中与MAPKs相互作用。实现这些目标的方法将基于重组DNA方法；western blotting, northern blotting，二维电泳，然后是MALDI-TOF质谱，免疫细胞化学，免疫沉淀，pull-down试验和激酶试验。预计该项目将显著推进细胞渗透感觉信号转导的知识。为渗透感觉信号转导的研究提供了一种新的泛盐鱼模型。广盐鱼类有一个肾外运输上皮，鳃上皮直接暴露在外部环境中，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、即时地进行操纵。本研究将对14-3-3的渗透调控提供全面的认识。A是控制渗透感觉信号转导和适应盐度变化的硬骨鱼鳃上皮细胞分化的许多方面的优秀候选分子。

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.