Signal Transduction in T Lymphocyte Activation

描述（由申请人提供）：PKC的重要性？TCR信号控制T细胞的激活和分化已经得到了很好的证实。PKC的选择性要求？在过敏和自身免疫性疾病中，而不是抗病毒反应中，突出了它作为一个有前途的药物靶点。实现这一潜力需要清楚地了解调节PKC独特，非冗余功能和高度选择性免疫突触（IS）定位的机制。在T细胞中，这是未知的。我们在上一个资助期间的工作表明，在PKC大家庭中，PKC？在调节这些事件的机制方面代表了一种独特的范式，并最终导致关键转录因子的激活，这些转录因子决定了T细胞的命运。我们建议采用多学科方法来解决以下未解决的问题：目标1)PKC的正调控？激活和定位。利用生物化学、遗传学、影像学和生物物理学的方法，我们将讨论PKC？由于其调节C1和V3结构域的独特性质，在T细胞中受到专门机制和质膜相互作用的积极调节。我们将对PKC的C1A、C1B和V3结构域进行详细的结构功能分析。它们的磷酸化，以及它们与结合伙伴的相互作用。目的2)PKC的自抑制作用C2域。我们的研究表明PKC？N端C2结构域负性调控其功能，tcr诱导的Tyr-90磷酸化减轻了自抑制作用，此外，C2本身是pTyr-和pip2结合结构域。我们将研究自抑制作用的机制，并表征c2结合配体及其功能意义。我们将与Wonhwa Cho博士（伊利诺伊大学）合作实现目标1和目标2，他将提供他在生物物理和双光子成像方法方面的专业知识，以进一步阐明调节PKC的专门机制。目的3)PKC对Ca2+/NFAT信号的调控。PKC吗?-/- T细胞显示Ca2+/NFAT信号缺陷，但连接PKC的机制？对Ca2+信号的影响是未知的。基于我们最近的发现，并考虑到Ca2+信号在决定生产性T细胞活化和能量/凋亡之间的平衡中的重要作用，我们将描述这一途径。具体来说，我们将讨论PKC？介导一个正反馈回路，通过其与Tec家族酪氨酸激酶的功能和/或物理关联，促进PLC31激活，从而促进Ca2+/NFAT信号传导。我们的研究将阐明控制PKC独特的膜定位和功能的新机制。在T细胞中这些新资料可能会为使用PKC提供合理的依据？作为自身免疫性和过敏性疾病以及T细胞恶性肿瘤的药物靶点。公共卫生相关性：蛋白激酶C-8 (PKC?)，一种在免疫系统的T淋巴细胞中表达的酶，对这些细胞的激活和存活以及它们建立许多有益和病理免疫反应的能力很重要，例如，防止某些感染，自身免疫和过敏性疾病。然而，控制PKC独特功能和细胞内定位的机制？在T淋巴细胞中（PKC家族的其他相关成员不共享），在很大程度上是未知的。在这里，我们建议使用一套互补的生化、遗传、成像和生物物理方法来阐明这些机制和PKC的调节相互作用。与其他蛋白质和膜脂一起。了解这些途径可能会为PKC的使用建立更合理的基础。作为自身免疫性和过敏性疾病以及T细胞白血病/淋巴瘤的药物靶点

DESCRIPTION (provided by applicant): The importance of PKC? in TCR signals controlling T cell activation and differentiation is well established. The selective requirement of PKC? in allergic and autoimmune diseases, but not in antiviral responses, highlights it as a promising drug target. Realization of this potential requires a clear understanding of the mechanism(s) that regulate the unique, non-redundant functions and highly selective immunological synapse (IS) localization of PKC? in T cells, which remain unknown. Our work during the last funding period revealed that within the large PKC family, PKC? represents a unique paradigm with respect to the mechanisms that regulate these events and, ultimately, lead to the activation of critical transcription factors, which determine T cell fate. We propose to apply a multi-disciplinary approach in order to address the following unresolved questions: Aim 1) Positive regulation of PKC? activation and localization. Using biochemical, genetic, imaging and biophysical approaches, we will address the hypothesis that the localization and activation of PKC? in T cells are positively regulated by specialized mechanisms and plasma membrane interactions owing to the unique properties of its regulatory C1 and V3 domains. We will conduct a detailed structure- function analysis of the C1A, C1B and V3 domains of PKC?, their phosphorylation, and their interaction with binding partners. Aim 2) Autoinhibition by the PKC? C2 domain. Our studies indicate that the PKC? N- terminal C2 domain negatively regulates its function, and that autoinhibition is relieved by TCR-induced phosphorylation of Tyr-90, and, furthermore, that C2 itself is a pTyr- and PIP2-binding domain. We will investigate the mechanisms through which autoinhibition is effected, and characterize C2-binding ligands and their functional significance. We will pursue Aims 1 and 2 as a collaborative consortium with Dr. Wonhwa Cho (U. Illinois), who will provide his expertise in biophysical and two-photon imaging approaches to further elucidate the specialized mechanisms that regulate PKC?. Aim 3) Regulation of Ca2+/NFAT signaling by PKC?. PKC?-/- T cells display a Ca2+/NFAT signaling defect, but the mechanism linking PKC? to Ca2+ signaling is unknown. Based on our recent findings, and given the important role of Ca2+ signaling in determining the balance between productive T cell activation and anergy/apoptosis, we will characterize this pathway. Specifically, we will address the hypothesis that PKC? mediates a positive feedback loop that promotes PLC31 activation and, hence, Ca2+/NFAT signaling, via its functional and/or physical association with Tec family tyrosine kinase(s). Our studies will elucidate novel mechanisms that control the unique membrane localization and function of PKC? in T cells. The new information will likely provide a rational basis for using PKC? as a drug target in autoimmune and allergic diseases, and in T cell malignancies. PUBLIC HEALTH RELEVANCE: Protein kinase C-8 (PKC?), an enzyme expressed in T lymphocytes of the immune system, is important for the activation and survival of these cells and their ability to mount many beneficial as well as pathological immune responses, e.g., protection against certain infections, and autoimmune and allergic diseases. However, the mechanisms, which control the unique functions and intracellular localization of PKC? in T lymphocytes (which are not shared by other related members of the PKC family), are largely unknown. Here we propose to use a complementary set of biochemical, genetic, imaging and biophysical approaches in order to elucidate these mechanisms and the regulatory interactions of PKC? with other proteins and membrane lipids. Understanding of these pathways is likely to establish a more rational basis for the use of PKC? as a drug target in autoimmune and allergic diseases, and in T cell leukemias/lymphomas