Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion.
E-selectin, a selectin expressed on activated vascular endothelium, supports rolling and stable adhesion of leukocytes at sites of inflammation. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-selectin with cytoskeletal elements, suggesting that outside-in signaling may occur during E-selectin-mediated adhesion. To investigate this potential signaling function of E-selectin, HUVEC activated with recombinant human IL-1beta (10 U/ml, 4 h) were labeled with [32P]orthophosphate, and E-selectin was immunoprecipitated using mAb H18/7. Autoradiography revealed constitutive phosphorylation of E-selectin in these cells and time-dependent dephosphorylation following adhesion of HL-60 cells. Cross-linking of cell surface E-selectin using H18/7 and a polyclonal secondary Ab induced E-selectin dephosphorylation, as did adhesion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand. Using adenoviral vector-mediated transfection in HUVEC of a tail-less E-selectin and phosphoamino acid analysis, we documented phosphorylation occurring exclusively within the cytoplasmic domain and involving serine residues. Additional experiments using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phosphorylated. Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin. Thus, E-selectin, which is constitutively phosphorylated in cytokine-activated human endothelial cells, undergoes an enzymatically regulated dephosphorylation following leukocyte adhesion. This process appears to be triggered by multivalent ligand binding and/or cross-linking of cell surface E-selectin. Ligand-dependent regulation of the phosphorylation of E-selectin's cytoplasmic domain provides additional evidence for a transmembrane signaling function of this molecule during leukocyte-endothelial interactions.
Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
M
M
Yoshida
B E
BE
Szente
J M
JM
Kiely
A
A
Rosenzweig
M A
MA
Gimbrone
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003432:Cross-Linking Reagents; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism*; D019040:E-Selectin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D008863:Microspheres; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D019040:E-Selectin; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D010768:Phosphoserine; D017027:Protein Tyrosine Phosphatases