Abstract
Na-K-Cl cotransport activity in duck erythrocytes increases approximately 10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires approximately 5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 approximately 0.7 microM). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase.
Highlights
Reversible phosphorylation of the cotransport protein, regulatory subunits, or upstream signal transducers [8, 9]
While cotransport activity appears to determined by a dynamic competition between ongoing protein kinase and phosphatase activities, a key question is whether all modes of stimulation involve phosphorylation of the cotransport protein itself and whether different stimuli involve different kinases
The recent advent of monoclonal antibodies capable of immunoprecipitating the Na-K-Cl cotransport protein from detergent extracts of 32P-labeled duck erythrocytes with high efficiency [26] makes it possible to quantitatively compare the phosphorylation induced by different stimuli and to assess the physical disposition of phosphorylation sites
Summary
Reversible phosphorylation of the cotransport protein, regulatory subunits, or upstream signal transducers [8, 9]. Duck erythrocytes have long served as a premier model of electroneutral ion transport by virtue of their simplicity, uniformity, and ease of experimental manipulation These cells manifest robust Na-K-Cl cotransport in response to four types of stimuli: osmotic cell shrinkage, elevated cytosolic cAMP (norepinephrine), Ser-Thr phosphatase inhibitors (calyculin-A, okadaic acid, endothall thioanhydride, fluoride), and deoxygenation [24, 25]. The recent advent of monoclonal antibodies capable of immunoprecipitating the Na-K-Cl cotransport protein from detergent extracts of 32P-labeled duck erythrocytes with high efficiency [26] makes it possible to quantitatively compare the phosphorylation induced by different stimuli and to assess the physical disposition of phosphorylation sites The results of this analysis suggest that all forms of activation promote phosphorylation of the cotransport protein at a common set of Ser/Thr sites.
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