Skubitz K M, Ahmed K, Campbell K D, Skubitz A P
Department of Medicine, University of Minnesota Medical School, Minneapolis.
J Immunol. 1995 Mar 15;154(6):2888-95.
CD50 (ICAM-3) is expressed at a high level on resting blood granulocytes, monocytes, and lymphocytes. The constitutive high expression of CD50 on resting leukocytes suggests that it is an important LFA-1 ligand in the initiation of the immune/inflammatory response. Using a radiolabeling technique initially designed to detect ecto-protein kinase activity, we found that CD50 mAbs immunoprecipitated a approximately 125- to 170-kDa phosphoprotein from human neutrophils. Phosphorylation was increased after stimulation with the chemotactic agent FMLP, platelet-activating factor, 12-O-tetradecanoyl-phorbol-13-acetate, and the calcium ionophore A23187. This increase in phosphorylation was transient with the maximal phosphorylation, being observed by 1 min. Phosphoamino acid analysis revealed that CD50 contained predominantly phosphotyrosine. Although this assay system was designed initially to detect ecto-protein kinase activity, subsequent studies have shown that membrane proteins can be phosphorylated on the cytoplasmic domain under these conditions. When CD50 was immunoprecipitated from solubilized neutrophils, protein tyrosine kinase activity associated with CD50 was detected in the immunoprecipitate. The data suggest that phosphorylation of CD50 on tyrosine by an associated tyrosine kinase plays a role in the function of CD50.
CD50（细胞间黏附分子-3）在静息血液中的粒细胞、单核细胞和淋巴细胞上高水平表达。CD50在静息白细胞上的组成性高表达表明它是免疫/炎症反应起始过程中一种重要的淋巴细胞功能相关抗原-1（LFA-1）配体。使用最初设计用于检测胞外蛋白激酶活性的放射性标记技术，我们发现CD50单克隆抗体从人中性粒细胞中免疫沉淀出一种约125至170 kDa的磷蛋白。在用趋化剂N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（FMLP）、血小板活化因子、12-O-十四烷酰佛波醇-13-乙酸酯（TPA）和钙离子载体A23187刺激后，磷酸化增加。这种磷酸化增加是短暂的，在1分钟时观察到最大磷酸化。磷酸氨基酸分析表明CD50主要含有磷酸酪氨酸。尽管该检测系统最初设计用于检测胞外蛋白激酶活性，但随后的研究表明在这些条件下膜蛋白的胞质结构域可以被磷酸化。当从溶解的中性粒细胞中免疫沉淀CD50时，在免疫沉淀物中检测到与CD50相关的蛋白酪氨酸激酶活性。数据表明，相关酪氨酸激酶对CD50酪氨酸的磷酸化在CD50的功能中起作用。
