Activation of the avian erythrocyte Na-K-Cl cotransport protein by cell shrinkage, cAMP, fluoride, and calyculin-A involves phosphorylation at common sites

Abstract

Na-K-Cl cotransport activity in duck erythrocytes increases ~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 ~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 ~ 0.7 μM). 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.