CD45 AND ITS ISOFORMS IN T CELL ACTIVATION

描述：（改编自研究者摘要）： 跨膜蛋白酪氨酸磷酸酶在T细胞 通过调节Lck和Fyn蛋白酪氨酸激酶的活性激活。 然而，CD 45功能的关键方面仍然没有得到解决。例如，CD 45 由多种亚型组成，不同之处仅在于它们的细胞外 域.这些差异分布在具有以下特征的T细胞亚群上： 不同的功能和激活要求。虽然实际作用的 个别亚型仍不清楚，已提出CD 45 细胞外结构域通过细胞质差异调节信号传导 域这就提出了CD 45是否有其他底物的问题。 此外，两个胞质结构域在调节酶活性中的作用 并且底物特异性不清楚。以往表达困难 已经克服了同一细胞系中不同的单个CD 45同种型 使用独特的模型，其中Jurkat人T细胞中的内源性CD 45表达 细胞白血病系已被特异性阻断转染的一个 CD 45反义基因。这样的CD 45-细胞已经被重建， 表达最大或最小的人CD 45同种型，或各种CD 45 PTB突变体。该模型使我们能够证明CD 45亚型特异性 IL-2分泌和酪氨酸磷酸化的差异， 几种关键的信号分子，包括zeta、ZAP-70、Vav和SLP-76。 此外，用含有特异性CD 45的CD 45-细胞转染这些CD 45-细胞， PTB结构域突变（包括底物捕获形式） 证明PTCD 4结构域2对于CD 45与 包括TCR ζ链在内的几种新型底物， ZAP-70的磷酸化作用。根据我们的调查结果，本申请旨在 为CD 45在T细胞活化中的作用提供了新的认识。这将 包括定义CD 45在调节这些新的信号传导中的作用， 底物和确定不同的CD 45亚型 差异调节T细胞活化。 在目标1中，我们将定义 CD 45胞质结构域与这些新底物的结合。在目标2中，我们 确定不同的CD 45细胞外结构域的表达 对应于最大（CD 45（ABC））和最小（CD 45（0））同种型 导致产生IL-2的能力改变;关注差异 调节近端事件（包括PTK活性和zeta磷酸化） 以及Vav和SLP-76，它们调节参与激活细胞的关键途径。 IL-2启动子。这些研究将为T细胞免疫学提供基础知识 激活信号和生长控制。因此，他们最终应该 提供对血液恶性肿瘤、免疫介导的 疾病和宽容。

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.