CD45 AND ITS ISOFORMS IN T CELL ACTIVATION

CD45 及其异构体在 T 细胞激活中的作用

• 批准号: 6373404
• 负责人: DAVID M ROTHSTEIN
• 金额: $ 26.07万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373404
• 关键词: CD antigens; T cell receptor; T lymphocyte; biological signal transduction; enzyme activity; human subject; interleukin 2; laboratory mouse; laboratory rabbit; leukocyte activation /transformation; phosphoprotein phosphatase; phosphorylation; protein biosynthesis; protein isoforms; protein tyrosine kinase; tissue /cell culture

DESCRIPTION: (Adapted from the Investigator's abstract): The CD45 family of transmembrane protein tyrosine phosphatases plays a key role in T cell activation by regulating the activity of Lck and Fyn protein tyrosine kinases. However, critical aspects of CD45 function remain unresolved. For example, CD45 is composed of multiple isoforms which differ only in their extracellular domains. These are differentially distributed on subsets of T cells having distinct functions and activation requirements. While the actual role of the individual isoforms remains unclear, it has been proposed that the CD45 extracellular domains differentially regulate signaling through the cytoplasmic domain. This raises the question as to whether CD45 has additional substrates. Moreover, the role of the two cytoplasmic domains in regulating enzyme activity and substrate specificity is unclear. Previous difficulties in expressing different individual CD45 isoforms in the same cell line have been overcome using a unique model whereby endogenous CD45 expression in the Jurkat human T cell leukemia line has been specifically blocked by transfection of a CD45-antisense gene. Such CD45- cells have been reconstituted to uniquely express either the largest o smallest human CD45 isoform, or various CD45 PTPase mutants. This model has allowed us to demonstrate CD45 isoform-specific differences in both IL-2 secretion and in the tyrosine phosphorylation of several key signaling moleules including zeta, ZAP-70, Vav, and SLP-76. Moreover, transfection of these CD45- cells with CD45 containing specific PTPase domain mutations (including substrate-trapping versions) has demonstrated that PTPase Domain 2 is critical for interaction of CD45 with several novel substrates including the TCR zeta chain, and is required for phosphorylatia n of ZAP-70. Based on our findings, this application seeks to provide new understanding of the role of CD45 in T cell activation. This will include defining the role of CD45 in regulating signaling through these new substrates and determining the mechanisms by which distinct CD45 isoforms differentially regulate T cell activation. In Aim 1 we will define the nature and consequences of the interaction of the CD45 cytoplasmic domain with these novel substrates. In Aim 2, we will determine how the expression of distinct CD45 extracellular domains corresponding to the largest (CD45(ABC ) and smallest (CD45(0)) isoforms results in altered capacity to produce IL-2; focusing on differential regulation of proximal events (including PTK activity and zeta phosphorylation) and Vav and SLP-76, that regulate key pathways involved in activation of the IL-2 promoter. These studies will provide fundamental knowledge about T cell activation signaling and growth control. As such, they should ultimately provide practical insight into hematological malignancie, immune-mediated diseases, and tolerance.

描述：（改编自研究者的摘要）：CD45 家族 跨膜蛋白酪氨酸磷酸酶在T细胞中起关键作用 通过调节 Lck 和 Fyn 蛋白酪氨酸激酶的活性来激活。 然而，CD45 功能的关键方面仍未解决。例如，CD45 由多种亚型组成，仅在细胞外不同 域。这些在具有以下特征的 T 细胞亚群上存在差异性分布： 不同的功能和激活要求。虽然实际作用 个别亚型仍不清楚，有人建议 CD45 细胞外结构域通过细胞质差异调节信号传导 领域。这就提出了 CD45 是否有额外底物的问题。 此外，两个细胞质结构域在调节酶活性中的作用 且底物特异性尚不清楚。以前表达困难 同一细胞系中不同的个体 CD45 同种型已被克服 使用独特的模型，Jurkat 人类 T 中内源性 CD45 表达 细胞白血病系已通过转染特异性阻断 CD45-反义基因。这种 CD45- 细胞已被重组为独特的 表达最大或最小的人类 CD45 同工型，或各种 CD45 PTP酶突变体。该模型使我们能够展示 CD45 亚型特异性 IL-2 分泌和酪氨酸磷酸化的差异 几个关键信号分子，包括 zeta、ZAP-70、Vav 和 SLP-76。 此外，用含有特定CD45的CD45-细胞转染这些CD45-细胞。 PTPase 结构域突变（包括底物捕获版本） 证明 PTPase 结构域 2 对于 CD45 与 几种新颖的底物，包括 TCR zeta 链，并且是 ZAP-70 的磷酸化。根据我们的发现，该应用程序旨在 为 CD45 在 T 细胞激活中的作用提供了新的认识。这将 包括通过这些新的定义 CD45 在调节信号传导中的作用 底物并确定不同 CD45 亚型的机制 差异性调节 T 细胞活化。 在目标 1 中，我们将定义相互作用的性质和后果 CD45 细胞质结构域与这些新底物。在目标 2 中，我们将 确定不同 CD45 胞外结构域的表达方式 对应于最大 (CD45(ABC)) 和最小 (CD45(0)) 同工型 导致产生 IL-2 的能力改变；注重差异化 近端事件的调节（包括 PTK 活性和 zeta 磷酸化） 以及 Vav 和 SLP-76，调节参与激活的关键途径 IL-2启动子。这些研究将提供有关 T 细胞的基础知识 激活信号传导和生长控制。因此，他们最终应该 提供对免疫介导的血液恶性肿瘤的实用见解 疾病和耐受性。