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 either osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, phosphorylation of the transporter occurs at a common set of serine/threonine 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 32P into the transporter and uptake of 86Rb by the cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in this manner, the activating kinase was 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine or fluoride. The inhibitory effect of swelling on kinase activity was progressively relieved by calyculin A, suggesting that the kinase itself is switched on by phosphorylation. The kinetics of activation by calyculin A conformed to an autocatalytic model in which the volume-sensitive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).
Lytle
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Division of Biomedical Sciences, University of California, Riverside 92521, USA.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004372:Ducks / Q000097:blood; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine / Q000494:pharmacology
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D028021:Sodium-Potassium-Chloride Symporters; C059041:calyculin A; D011494:Protein Kinases; D019311:Staurosporine; D005033:Ethylmaleimide