Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells.
The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.
Wang
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KK
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Du
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Diglio
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Tsang
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Kuo
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D000818:Animals; D001013:Aorta, Thoracic; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006728:Hormones / Q000494:pharmacology*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D006728:Hormones; D010750:Phosphoproteins; D013873:Thionucleotides; D010769:Phosphothreonine; D010768:Phosphoserine; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium