Regulation of erythrocyte Na-K-2Cl cotransport by threonine phosphorylation.
A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.
Membrane Biology Group, College of Medicine and Veterinary Medicine, School of Biomedical and Clinical Laboratory Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, UK.
Ioulia
I
Matskevich
Karen L
KL
Hegney
Peter W
PW
Flatman
D000818:Animals; D004912:Erythrocytes / Q000378:metabolism*; D005289:Ferrets; D019833:Genistein / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008387:Marine Toxins; D052998:Microcystins; D008970:Molecular Weight; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry*; D054645:Protein Phosphatase 1; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000097:blood*; D019311:Staurosporine / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D008387:Marine Toxins; D052998:Microcystins; D010080:Oxazoles; D010456:Peptides, Cyclic; D011720:Pyrazoles; D011743:Pyrimidines; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D010769:Phosphothreonine; D013912:Threonine; C078588:microcystin; C059041:calyculin A; D019833:Genistein; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D019311:Staurosporine; D008274:Magnesium