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

该项目的总体目标是检验磷酸蛋白接头 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。 具体来说，该项目将研究 14-3-3.a 在多个水平（表达、翻译后调节和区室化）的渗透调节机制及其与丝裂原激活蛋白激酶（MAPK）在广盐鱼异斜鱼适应鳃上皮中的相互作用。 该项目的重点是对 14-3-3.a 的渗透调节进行详细研究。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且非凡的重要性。这些蛋白质是一种新型的分子接头，可调节涉及环境调节信号通路、细胞分化和细胞周期控制的几乎所有重要成分之间的相互作用（Fu et al., 2000）。 14-3-3 蛋白通过结合和隔离丝氨酸或苏氨酸磷酸化蛋白来调节细胞活性和功能。这些分子可能在广盐性硬骨鱼盐度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应信号转导的主要机制，2) 14-3-3 蛋白质参与许多有丝分裂、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。从广盐鱼 F.heteroclitus (GB AF302039) 中克隆并测序的新型 cDNA 包含一个开放阅读框，编码第一个已知的鱼类 14-3-3 蛋白，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 基因在从海水 (SW) 转移到淡水 (FW) 的异斜梭菌的鳃上皮中被强烈诱导。 从 SW 转移到 FW 的异斜鱼鳃上皮中 MAPK ERK、JNK 和 p38 的丰度和活性表明，在广盐鱼的盐度驯化过程中，所有 MAPK 的活性都受到强烈调节。 目的是详细分析广盐鱼鳃上皮中14-3-3.a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概况。 2) 研究渗透压变化如何影响 14-3-3.a 的翻译后修饰。 3) 了解14-3-3.a的细胞和亚细胞定位及其渗透压调节方式。 4) 确定14-3-3.a是否与渗透适应鳃上皮中的MAPK相互作用。 实现这些目标的方法将基于重组 DNA 方法；蛋白质印迹、北方印迹、二维电泳，然后进行 MALDI-TOF 质谱分析、免疫细胞化学、免疫沉淀、下拉分析和激酶分析。 预计该项目将显着推进细胞渗透感应信号转导的知识。 它将把一种新型的广盐鱼模型引入渗透感应信号转导研究。广盐鱼类具有肾外运输上皮，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且即时地控制。 这项研究应该为14-3-3.a的渗透调节提供全面的见解，14-3-3.a是一种优秀的候选分子，用于控制渗透感应信号转导和细胞分化的许多方面，以适应广盐性硬骨鱼的鳃上皮细胞暴露于盐度变化。

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.