The primary goal of this proposal is to determine how the plasma membrane (Na,K)-ATPase is regulated in friend virus transformed murine erythroleukemia cells. The secondary goals are to investigate other Na+ and Ca++ fluxes in plasma membranes from these cells. Friend cells may be grown indefinitely in suspension but undergo an apparently normal erythropoesis when treated with dimethyl sulfoxide (DMSO) or a variety of other agents. The interest in cation fluxes stems from the observation that ouabain (which specifically inhibits the (Na,K) ATPase from the outside of the cell) induced differentiation in some Friend cell lines and acts synergistically with other inducers to accelerate commitment in other cell lines. From studies of cation fluxes during the past two years we have developed a scheme to explain the early events essential in Frient cell commitment: 1) the inducer inhibits the (Na,K)ATPase 2) Cytoplasmic Na+ rises 3) Ca++ influx is accelerated probably via a Na+/Ca++ antiport and 4) Ca++ signals a series of events essential for commitment. The mechanism by which Ca++ acts is obscure but it may turn out to be a general signal for diferentiation in a variety of developing cells. While studying the plasma membranes from these cells we discovered a membrane bound kinase which specifically and stoichiometrically phosphorylates a threonine residue on the Alpha subunit of the (Na,K)ATPase. The Alpha subunit is also phosphorylated on a threonine residue in vivo and preliminary results suggest that the level of phosphorylation decreases with committment. The obvious implication that this kinase may be a mechanism for regulating the (Na,K)ATPase in a variety of cells in supported by our recent discovery of a similar (Na,K)ATPase specific kinase in plams membranes from shark rectal glan (a hormonally sensitive ion secreting tissue). Thus, initial efforts will focus on characterizing the kinase, determining whether it alters pumping by the (Na,K)ATPase, and determining how the kinase is controlled in vivo. It is hoped that this research will lead to an understanding of hormonal regulation of the (Na,K)ATPase and may also provide some understanding of how cells are maintained in a transformed state.
Showing the most recent 10 out of 18 publications
