SIGNAL TRANSDUCTION IN T-LYMPHOCYTE ACTIVATION

触发T细胞受体（TCR）/CD3复合物产生信号， 穿过细胞到达细胞核，导致基因转录，激活 和扩散。 阐明信号事件对于 了解T细胞在生理和病理（自身免疫， 恶性）反应。 我们以前的研究集中在磷脂酶C (PLC)- 介导的，第二信使产生途径，激活 蛋白激酶C（PKC）和动员Ca 2+。 它正在成为 然而，很明显，这一途径是在一个关键的，但很差的调节， 酪氨酸蛋白激酶（TPKs）是酪氨酸蛋白激酶（Tyrosine protein kinases，TPKs）的一种。 拟议 研究阐明了肌醇的作用及其相互作用， 磷脂和TPK信号通路。 第一，聚合酶 链式反应（PCR）和基因克隆技术将用于克隆和 表征编码T细胞表达的PKC亚型的cDNA，包括 初步研究显示的推定新亚型。 一种多探针RNA酶 保护试验将用于分析PKC基因的表达模式 在胸腺和外周T细胞亚群中以及在人T细胞活化期间。 第二，将采取以下方法阐明 两种T细胞表达的TPK，p59fyn和p56lck，与 TCR/CD3复合物或CD4/CD8共受体，分别在T细胞中， 信令，特别是他们可以调节PLC的模式 激活：1）这两个TPK的激活状态为 在TCR/CD3触发的T细胞中，通过以下步骤顺序分析： 两个保守的酪氨酸残基参与的磷酸化水平 TPK活性的正或负调节。 2)不 细胞将用设计成 选择性抑制LCK或Fyn基因的表达，使用反义 RNA和靶向核酶方法以及突变的， 不活跃的TPK。 这些实验操作对信号 转导事件（PLC激活、基因诱导和增殖）将 进行分析并与p56 LCK或p59 Fyn水平相关联。 3)建立 使用抗CD3抗体的TCR/CD3触发后磷酸化的TPK底物的"图谱" 磷酸酪氨酸抗体，尝试识别它们，并确定是否 肌醇磷脂水解通过酪氨酸与TPK活化偶联 PLC或与受体TPK相关的其他底物的磷酸化。 4)将平行分析各种TPK抑制剂对 p56lck或p59fyn活性，TCR/CD3诱导的酪氨酸磷酸化和 后来的信号事件。 5)活化缺陷型CD4-CD8-lpr T细胞或 它们的代表性T细胞杂交瘤，显示出组成型增加 酪氨酸磷酸化（p59fyn过表达的结果）和 磷酸肌醇水解，将作为一个可能的自然模型进行研究 表明酪氨酸磷酸化和PLC之间存在因果关系 activation. 了解PKC和TPKs在T细胞活化中的作用 可能导致合理设计药理学方法， 纠正由缺陷信号传导引起的异常T细胞应答。

Triggering the T cell receptor (TCR)/CD3 complex generates signals that traverse the cell to the nucleus, leading to gene transcription, activation and proliferation. Elucidation of signaling events is crucial to understand T cell behavior in physiological and pathological (autoimmune, malignant) responses. Our previous studies focused on a phospholipase C (PLC) - mediated, second messenger - generating pathway that activates protein kinase C (PKC) and mobilizes Ca2+, respectively. It is becoming clear, however, that this pathway is regulated in a critical, yet poorly understood, manner by tyrosine protein kinases (TPKs). The proposed studies address the roles of, and interaction between, the inositol phospholipid and TPK signaling pathways in T cells. First, polymerase chain reaction (PCR) and gene cloning techniques will be used to clone and characterize cDNAs encoding T cell-expressed PKC isoforms, including a putative new isoform revealed by preliminary studies. A multiprobe RNAse protection assay will be used to analyze expression patterns of PKC genes in thymic and peripheral T cell subsets and during human T cell activation. Second, the following approaches will be undertaken to elucidate the roles of two T cell-expressed TPKs, p59fyn and p56lck, that are associated with the TCR/CD3 complex or the CD4/CD8 coreceptors, respectively, in T cell signaling, in particular the mode by which they may regulate PLC activation: 1) The activation status of these two TPKs will be sequentially analyzed in TCR/CD3-triggered T cells by following the phosphorylation levels of two conserved tyrosine residues involved in positive or negative regulation of the TPK's activity, respectively. 2) T cells will be stably transfected with expression vectors designed to selectively inhibit the expression of the lck or fyn genes, using antisense RNA and targeted ribozyme approaches as well as expression of mutated, inactive TPKs. The effects of these experimental manipulations on signal transduction events (PLC activation, gene induction and proliferation) will be analyzed and correlated with p56lck or p59fyn levels. 3) Establish "maps" of TPK substrates phosphorylated upon TCR/CD3 triggering using anti- phosphotyrosine antibodies, attempt to identify them, and determine whether inositol phospholipid hydrolysis is coupled to TPK activation via tyrosine phosphorylation of PLC or other substrates associated with receptor TPKs. 4) Various TPK inhibitors will be analyzed in parallel for their effects on p56lck or p59fyn activity, TCR/CD3-induced tyrosine phosphorylation and later signaling events. 5) Activation-defective CD4-CD8- lpr T cells or their representative T cell hybridomas, that display increased constitutive tyrosine phosphorylation (a result of overexpressed p59fyn) and phosphoinositide hydrolysis, will be studied as a possible natural model manifesting a causal relationship between tyrosine phosphorylation and PLC activation. Understanding the roles of PKC and TPKs in T cell activation may lead to rational design of pharmacological approaches aimed at correcting abnormal T cell responses resulting from defective signaling.