A VOLUME-SENSITIVE PROTEIN-KINASE REGULATES THE NA-K-2CL COTRANSPORTER IN DUCK RED-BLOOD-CELLS

When Na-K-2Cl cotransport is activated in duck red blood cells by eith er osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, p hosphorylation of the transporter occurs at a common set of serine/thr eonine sites. To examine the kinetics and regulation of the activating kinase, phosphatase activity was inhibited abruptly with calyculin A and the subsequent changes in transporter phosphorylation and activity were determined. Increases in fractional incorporation of P-32 into t he transporter and uptake of Rb-86 by the cells were closely correlate d, suggesting that the phosphorylation event is rate determining in th e activation process. Observed in this manner, the activating kinase w as 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, sta urosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine o r fluoride. The inhibitory effect of swelling on kinase activity was p rogressively relieved by calyculin A, suggesting that the kinase itsel f is switched on by phosphorylation. The kinetics of activation by cal yculin A conformed to an autocatalytic model in which the volume-sensi tive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).