The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.
The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of
Pewitt
E B
EB
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
Hegde
R S
RS
Haas
M
M
Palfrey
H C
HC
D000818:Animals; D017487:Ankyrins; D001717:Birds; D001798:Blood Proteins / Q000378:metabolism; D002034:Bumetanide / Q000097:blood*; D002227:Carbazoles / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood*; D002712:Chlorides / Q000097:blood*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D026121:Indole Alkaloids; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D019319:Okadaic Acid; D010766:Phosphorylation; D011188:Potassium / Q000097:blood*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000097:blood*; D012964:Sodium / Q000097:blood*; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides
D017487:Ankyrins; D001798:Blood Proteins; D002227:Carbazoles; D002352:Carrier Proteins; D002712:Chlorides; D004988:Ethers, Cyclic; D026121:Indole Alkaloids; D007546:Isoquinolines; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; D002034:Bumetanide; D019319:Okadaic Acid; C044388:N-(2-aminoethyl)-5-isoquinolinesulfonamide; C049985:staurosporine aglycone; D012964:Sodium; D011494:Protein Kinases; D011188:Potassium