Identification of the kinases and phosphatases that regulate phosphorylation and activity of the Na-K-2Cl cotransporter in response to environmental change (360G-Wellcome-077711_Z_05_Z)

The Na-K-2Cl cotransporter plays important roles in regulating the compositionand volume of cells and body fluids. Its activity changes in response to diverse stimuli due to its phosphorylation at several sites. We will test the hypothesis that regulation of cotransporter activity involves phosphorylation of the cotransporter at multiple sites by several different kinases and phosphatases and aim to identify the enzymes and sites involved. We will use ferret red cells as a powerful functional model of mammalian cotransport, and cloned cotransporter expressed in HEK-293 cells so we can exploit molecular biological techniques. We will:· Identify proteins that co-precipitate with the cotransporter by mass spectroscopy following their isolation from red cells by blue native gel electrophoresis, co-immunoprecipitation, and pull-down assays using cotransporter with genetically-modified kinase and phosphatase binding sites.· Identify sites on the cotransporter that are phosphorylated by mass spectroscopy, 2D electrophoresis, and using sequence information from cloning studies.· Analyse how cotransporter activity