The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues.
P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.
Fujimoto
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W.K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
McEver
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000302:isolation & purification / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D010879:Piperazines / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins
D000470:Alkaloids; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D007546:Isoquinolines; D019007:P-Selectin; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D010879:Piperazines; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013917:Thrombin; D000527:Alprostadil; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate