PHOSPHORYLATION OF THE CYTOPLASMIC DOMAIN OF E-SELECTIN IS REGULATED DURING LEUKOCYTE-ENDOTHELIAL ADHESION

E-selectin, a selectin expressed on activated vascular endothelium, su pports rolling and stable adhesion of leukocytes at sites of inflammat ion. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-s electin with cytoskeletal elements, suggesting that outside-in signali ng may occur during E-selectin-mediated adhesion, To investigate this potential signaling function of E-selectin, HUVEC activated with recom binant human IL-1 beta (10 U/ml, 4 h) were labeled with [P-32]orthopho sphate, and E-selectin was immunoprecipitated using mAb H18/7, Autorad iography 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 pol yclonal secondary Ab induced E-selectin dephosphorylation, as did adhe sion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand, Using adenoviral vector-mediated tran sfection in HUVEC of a tail-less E-selectin and phosphoamino acid anal ysis, we documented phosphorylation occurring exclusively within the c ytoplasmic domain and involving serine residues. Additional experiment s using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phospho rylated, Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin, Thus, E-selectin, w hich is constitutively phosphorylated in cytokine-activated human endo thelial 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-sele ctin, 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 inte ractions.