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

TCR可变的a/b和g/d异源二聚体与不变的CD3 g、d、e和z蛋白相关，从而形成TCR/CD3复合物。TCR的激活信号由免疫球蛋白家族酪氨酸激活基序（ITAMs）转导，ITAMs将TCR与细胞蛋白和适配器分子偶联。CD3g，d和e链每个都包含一个ITAM，而CD3z链在其细胞质尾部包含三个ITAM。CD3g、d和e的itam中的酪氨酸残基在激活过程中被磷酸化。CD3e亚基在TCR信号转导的启动中起着重要作用。抗cd3e单克隆抗体的刺激在未成熟胸腺细胞中引起细胞凋亡，而在成熟T细胞中引起活化、能量和凋亡。已发表的研究表明，可能存在两种不同的死亡信号传导途径：一种由CD3e触发，另一种由CD3z触发。CD3e启动的细胞凋亡信号通路尚不清楚。为了解决CD3e异常信号在T细胞凋亡中的作用，构建了一个嵌合分子（称为CD8e），将人CD8的细胞外和跨膜结构域融合到CD3e的细胞质结构域，并产生了突变体Y171F、Y180F和Y171F/Y180F。这些DNA构建物被稳定地引入Jurkat白血病CD8细胞。抗cd8抗体刺激显示，野生型CD3e表达细胞发生凋亡，而突变细胞不发生凋亡，这表明这两个酪氨酸残基是CD3e诱导的白血病细胞凋亡信号转导所必需的。酪氨酸磷酸化CD3e导致磷脂酰肌醇3'-激酶（pi3 -激酶）p85a亚基募集，提示PI3K/AKT依赖的信号通路在细胞凋亡调控中的作用。TCR/CD3复合物的刺激也会导致Ras-Raf-Mek-Erk级联的激活。我们之前已经证明，Raf-1在防止细胞凋亡中作为效应激酶的Cl1-2起作用。为了进一步确定这些途径对CD3e凋亡信号传导的作用，我们将利用表达野生型（能诱导凋亡）或突变型CD3e受体（不能诱导凋亡）的Jurkat细胞系，并分析它们对信号通路的利用。了解CD3e刺激后死亡或存活的机制将有助于设计治疗白血病的新治疗方法。

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.