Regulation of the activity and phosphorylation of the plasma membrane Ca(2+)-ATPase by protein kinase C in intact human erythrocytes.
Previously the plasma membrane-bound or purified Ca(2+)-translocation ATPase (Ca2+ pump) was found to be activated and phosphorylated by protein kinase C in vitro (K. K. W. Wang et al. 1991, J. Biol. Chem. 266, 9078-9085). We now show that in intact human erythrocytes phorbol-12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, decreases the amplitude of the intracellular calcium ([Ca2+]i) transient induced by 2.5 microM CaCl2 and 10 microM A23187. Since PMA did not affect Ca2+ influx, the decrease in amplitude was most likely due to the stimulation of the Ca2+ pump, the major mechanism of calcium extrusion in these cells. The effect was dose-dependent, the maximum decrease in amplitude (33%) occurring at 1 microM PMA. The depression of the [Ca2+]i transient was further enhanced by the phosphatase inhibitor okadaic acid. It was reversed by the protein kinase C inhibitor staurosporine and could not be mimicked by inactive PMA analogues. In erythrocytes labeled with [32P]orthophosphate, PMA treatment phosphorylated the Ca(2+)-ATPase in a dose-dependent manner. The phosphorylation was inhibited by staurosporine and was slightly enhanced by okadaic acid. Changes in lipid phosphorylation and content were studied under the same conditions in intact cells. The turnover of 32P and lipid phosphate in phosphatidylinositol 4,5-bisphosphate (PIP2) was inhibited by 1 mM adriamycin, concomitant with an increased amplitude of the [Ca2+]i transient. The PIP2 content and its 32P radioactive did not, however, change with PMA stimulation. We conclude that while both protein kinase C and polyphosphoinositides are regulators of Ca(2+)-ATPase activity in the intact human erythrocyte, stimulation of the enzyme activity by PMA is predominantly protein kinase C-mediated.
Wright
L C
LC
Department of Pharmacy, University of Sydney, NSW, Australia.
Chen
S
S
Roufogalis
B D
BD
D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008563:Membrane Lipids / Q000097:blood; D010710:Phosphates / Q000097:blood; D010743:Phospholipids / Q000097:blood; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008563:Membrane Lipids; D010710:Phosphates; D010743:Phospholipids; D004317:Doxorubicin; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium