Yoshida M, Szente B E, Kiely J M, Rosenzweig A, Gimbrone M A
Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
J Immunol. 1998 Jul 15;161(2):933-41.
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.
E选择素是一种在活化的血管内皮细胞上表达的选择素，可支持白细胞在炎症部位的滚动和稳定黏附。此前，我们曾报道白细胞与培养的内皮细胞黏附会诱导E选择素的胞质结构域与细胞骨架成分发生关联，这表明在E选择素介导的黏附过程中可能发生由外向内的信号传导。为了研究E选择素的这种潜在信号功能，用重组人白细胞介素-1β（10 U/ml，4小时）激活的人脐静脉内皮细胞（HUVEC）用[32P]正磷酸盐标记，并用单克隆抗体H18/7免疫沉淀E选择素。放射自显影显示这些细胞中E选择素的组成性磷酸化以及HL-60细胞黏附后的时间依赖性去磷酸化。使用H18/7和多克隆二抗对细胞表面E选择素进行交联可诱导E选择素去磷酸化，用重组P选择素糖蛋白配体-1（PSGL-1，一种E选择素配体）包被的珠子黏附也可诱导其去磷酸化。通过腺病毒载体介导在HUVEC中转染无尾E选择素并进行磷酸氨基酸分析，我们证明磷酸化仅发生在胞质结构域内且涉及丝氨酸残基。使用在COS-7细胞中表达的一系列E选择素胞质结构域突变体进行的额外实验确定了组成性磷酸化的区域。用冈田酸和钒酸钠预孵育可消除黏附诱导的E选择素去磷酸化。因此，在细胞因子激活的人内皮细胞中组成性磷酸化的E选择素在白细胞黏附后会经历酶促调节的去磷酸化。这个过程似乎是由多价配体结合和/或细胞表面E选择素的交联触发的。E选择素胞质结构域磷酸化的配体依赖性调节为该分子在白细胞-内皮细胞相互作用期间的跨膜信号功能提供了额外证据。
