SIGNAL TRANSDUCTION IN T-LYMPHOCYTE ACTIVATION

触发T细胞受体(TCR)/CD3复合体产生的信号 穿过细胞到达细胞核，导致基因转录、激活 和扩散。对信号事件的解释对于 了解T细胞在生理和病理方面的行为(自身免疫、 恶性)反应。我们之前的研究集中在磷脂酶C (PLC)介导的第二信使生成途径，激活 蛋白激酶C(PKC)和钙离子的动员。它正在成为 然而，清楚的是，这一途径受到了一种关键但很差的调控 理解，方式由酪氨酸蛋白激酶(TPKs)。建议数 研究涉及肌醇的作用和相互作用 T细胞中的磷脂和TPK信号通路。第一，聚合酶 将利用链式反应(PCR)和基因克隆技术克隆和 鉴定编码T细胞表达的PKC亚型的cDNA，包括 初步研究发现了推测的新的同工酶。一种多探针核糖核酸酶 保护实验将用于分析PKC基因的表达模式 在胸腺和外周T细胞亚群中以及在人类T细胞激活期间。 第二，将采取以下方法来阐明这些角色 两个T细胞表达的TPKs，p59fyn和p56lck，与 T细胞中的TCR/CD3复合体或CD4/CD8共受体 信令，特别是它们调节PLC的方式 激活：1)这两个TPK的激活状态为 通过以下方式在TCR/CD3触发的T细胞中进行顺序分析 两个保守的酪氨酸残基参与的磷酸化水平 对TPK活性的正向或负向调节。2)T 细胞将稳定地被设计成能够 利用反义技术选择性抑制lck或fyn基因的表达 RNA和靶向核酶方法以及突变的、 非活动的TPK。这些实验操作对信号的影响 转导事件(PLC激活、基因诱导和增殖)将 与p56lck或p59fyn水平进行相关分析。3)建立 TCR/CD3激活的TPK底物的“MAP” 磷酸酪氨酸抗体，试图识别它们，并确定 肌醇磷脂的水解与酪氨酸激活的TPK偶联 PLC或与受体TPKs相关的其他底物的磷酸化。 4)将并行分析不同的TPK抑制剂对 P56lck或p59fyn活性，TCR/CD3诱导的酪氨酸磷酸化和 稍后的信号事件。5)激活缺陷的CD4-CD8-LPR T细胞或 他们的代表T细胞杂交瘤，表现出更多的结构性 酪氨酸磷酸化(p59fyn过表达的结果)和 肌醇磷脂的水解，将作为一种可能的自然模型进行研究 酪氨酸磷酸化与PLC之间的因果关系 激活。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，目的是 纠正由信号缺陷引起的异常T细胞反应。

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.