Apoptosis Signaling in T Lymphocyte Leukemia Cells Induced by the TCR/CD3 Complex

TCR/CD3 复合物诱导 T 淋巴细胞白血病细胞的凋亡信号转导

• 批准号: 5222944
• 负责人: Professor Dr. Ulf Rüdiger Rapp
• 金额: --
• 依托单位: National Laboratory of Medical Molecular Biology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2000
• 资助国家: 德国
• 起止时间: 1999-12-31 至 2003-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5222944?language=en
• 关键词: Apoptosis; Signaling; Lymphocyte; Leukemia; Cells

TCR variable heterodimers of a/b and g/d are associated with invariable CD3 g,d,e and z proteins, thus forming the TCR/CD3 complex. The activation signals of TCR are transduced by immunoglobulin family tyrosine-based activation motifs (ITAMs), which couple the TCR to cellular proteins and adapter molecules. CD3g,d and e each contain a single ITAM whereas CD3z chain contains three in its cytoplasmic tail. The tyrosine residues in the ITAMs of CD3g,d and e are phosphorylated during activation. The CD3e subunit plays an important role in the initiation of TCR signal transduction. Stimulation by anti-CD3e monoclonal antibodies causes cell death by apoptosis in immature thymocytes, but activation, anergy and apoptosis in mature T cells. Published work suggests that there might be two different death-signaling pathways: one triggered by CD3e and another by CD3z. The apoptosis signaling pathway initiated by CD3e is poorly understood. To address the role of aberrant signaling through CD3e in T cell apoptosis a chimeric molecule (termed CD8e) that fuses the extracellular and transmembrane domains of human CD8 to the cytoplasmic domain of CD3e was constructed and the mutants Y171F, Y180F and Y171F/Y180F were generated. These DNA contructs were stably introduced into Jurkat leukemia CD8 cells. Stimulation with anti-CD8 antibody showed that apoptosis occurred in the wild type CD3e expressing cells but not in the mutant cells, suggesting that the two tyrosine residues are required for CD3e-induced apoptotic signaling in leukemia cells. Tyrosine phosphorylation of CD3e resulted in the recruitment of the phosphatidylinositol 3'-kinase (PI3-kinase) p85a subunit iundicating a role for PI3K/AKT dependent signaling pathways in apoptosis regulation. Stimulation of the TCR/CD3 complex also results in the activation of the Ras-Raf-Mek-Erk cascade. We have previously demonstrated that Raf-1 functions as effector kinase for Cl1-2 in preventing apoptosis. To further define the role of these pathways for apoptosis signaling by CD3e we will utilize Jurkat cell lines expressing wild type (competent for apoptosis induction) or mutant forms of the CD3e receptor (defective for apoptosis induction) and analyze them for signaling pathway utilization. Understanding of the mechanisms governing death or survival after CD3e stimulation will allow for the design of new therapeutic approaches for the treatment of leukemia.

A/b和g/d的TCR可变异源二聚体与恒定的CD3g、d、e和z蛋白结合，形成TCR/CD3复合体。TCR的激活信号由基于酪氨酸的免疫球蛋白家族激活基序(ITAM)传递，免疫球蛋白家族酪氨酸激活基序将TCR与细胞蛋白质和接头分子偶联。CD3g、d和e各含有一个ITAM，而CD3z链在其胞质尾部含有三个。CD3g、d和e的ITAM中的酪氨酸残基在激活过程中被磷酸化。CD3e亚单位在启动TCR信号转导中起重要作用。在抗CD3e单抗的刺激下，未成熟胸腺细胞通过凋亡导致细胞死亡，而成熟T细胞则通过激活、无能和凋亡引起细胞死亡。发表的研究表明，可能存在两种不同的死亡信号通路：一种是由CD3e触发的，另一种是由CD3z触发的。CD3e启动的细胞凋亡信号转导通路知之甚少。为探讨CD3e异常信号在T细胞凋亡中的作用，构建了融合人CD8胞外区和跨膜区与CD3e胞内区的嵌合分子(CD8e)，并产生了突变体Y171F、Y180F和Y171F/Y180F。这些DNA载体被稳定地导入Jurkat白血病CD8细胞。经抗CD8抗体刺激后，野生型CD3e表达细胞发生了凋亡，而突变型细胞未发生凋亡，提示这两个酪氨酸残基是CD3e诱导白血病细胞凋亡信号所必需的。CD3e的酪氨酸磷酸化导致磷脂酰肌醇3‘-激酶(PI3-Kinase)p85a亚单位的募集，提示PI3K/AKT依赖的信号通路在细胞凋亡调控中发挥作用。刺激TCR/CD3复合体也会激活Ras-Raf-MEK-Erk级联。我们先前已经证明Raf-1作为CL1-2的效应蛋白在阻止细胞凋亡中发挥作用。为了进一步明确这些途径在CD3e诱导的细胞凋亡信号中的作用，我们将利用表达CD3e受体野生型(可诱导凋亡)或突变形式(不能诱导凋亡)的Jurkat细胞株，并对它们进行信号通路利用分析。了解CD3e刺激后死亡或存活的机制将有助于设计新的治疗白血病的方法。