Animal cells must counteract osmotic stresses which continually threaten their volume and integrity. A major goal is to understand how animal cells sense perturbations in their fluid volume and then activate membrane transporters that restore cell solute and water content. Volume appears to be detected by a mechanism that transmits information on intracellular macromolecular crowding and ionic strength to separate shrinkage-activated and swelling-activated transporters via protein phosphorylation. One prominent mechanism for volume recovery after cell shrinkage is Na-K-2Cl cotransport (NKCC). The NKCC-1 membrane transporter mediates salt and water uptake when phosphorylated by a poorly characterized Ser/Thr protein kinase called CT-kinase. Seconds after cell shrinkage, CT-kinase is activated, apparently when phosphorylated by another kinase of unknown identity. As a first step towards understanding this signaling system, CT-kinase will be isolated, and probes based on its peptide sequence will be used to identify cDNA clones from mouse 3T3 cells. Fusion proteins and antibodies will then be generated to facilitate studies on the requirements for activation of CT-kinase in intact cells. The role of CT-kinase in cell volume perception and regulation will be evaluated in cells overexpressing active or inactive forms of the enzyme. The effect of macromolecular crowding agents, ionic strength, and chloride concentration on CT-kinase activity and phosphorylation will be tested using in vivo and in vitro experimental systems. A central aim is to evaluate the unifying theory that CT-kinase coordinately regulates both shrinkage-activated and swelling-activated ion transporters. While the focus of this project will be CT-kinase and its regulation of NKCC1, the findings are likely to prove applicable to other protein kinases important in cellular responses to osmotic stress.
