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

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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.