Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl−cotransporter

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Abstract
Previous studies from our laboratory have shown a close correlation between increased Na+-K+-2Cl−cotransporter activity and increased cotransporter phosphorylation after β-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We also show that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that β-adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. In addition, we demonstrate that the increased phosphorylation on the cotransporter resulting from β-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western blotting experiments demonstrate that this peptide is derived from the NH2-terminal cytosolic tail of the cotransporter, which surprisingly does not contain the sole protein kinase A consensus site on the molecule.