Signal Transduction in T Lymphocyte Activation

描述(由申请人提供)：PKC的重要性？在TCR中，控制T细胞激活和分化的信号被很好地建立起来。PKC的选择性要求？在过敏和自身免疫性疾病中，而不是在抗病毒反应中，突出了它作为一个有前途的药物靶点的前景。要实现这一潜力，需要清楚地理解调控蛋白激酶C的独特、非冗余功能和高选择性免疫突触定位的机制(S)。在T细胞中，这仍然是未知的。我们在上一个资助期的工作表明，在PKC大家庭中，PKC？代表了一种独特的机制，调节这些事件，并最终导致关键转录因子的激活，这决定了T细胞的命运。我们建议采用多学科的方法来解决以下悬而未决的问题：目标1)积极监管PKC？激活和本地化。利用生化、遗传学、成像学和生物物理学的方法，我们将解决这样的假设：PKC？在T细胞中，由于其调节的C1和V3结构域的独特性质，其正向调节机制和质膜相互作用是积极的。我们将对PKC？的C1a、C1B和V3结构域、它们的磷酸化以及它们与结合伙伴的相互作用进行详细的结构和功能分析。目的2)PKC的自身抑制作用？C2结构域。我们的研究表明，PKC？N-末端的C2结构域负向调节其功能，这种自我抑制通过TCR诱导的Tyr-90的磷酸化而解除，而且C2本身是一个pTyr和PIP2结合域。我们将研究影响自身抑制的机制，并表征C2结合配体及其功能意义。我们将与伊利诺伊州大学的Wonhwa Cho博士合作，致力于目标1和目标2，后者将提供他在生物物理和双光子成像方法方面的专业知识，以进一步阐明监管PKC的专门机制？目的3)PKC？对钙/NFAT信号的调节作用。PKC？-/-T细胞存在钙离子/NFAT信号缺陷，但连接PKC？-/-T细胞的机制不同。对Ca~(2+)信号转导未知。根据我们最近的发现，并考虑到钙信号在决定生产性T细胞激活和无能/凋亡之间的平衡方面的重要作用，我们将对这一途径进行描述。具体地说，我们将解决PKC？通过与Tec家族酪氨酸激酶(S)的功能和/或物理联系，介导促进PLC31激活的正反馈环路，从而促进钙/NFAT信号转导。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。新的信息可能会为使用PKC提供一个合理的基础。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。与公众健康相关：蛋白激酶C-8(PKC？)是一种在免疫系统的T淋巴细胞中表达的酶，对于这些细胞的激活和生存以及它们启动许多有益的和病理的免疫反应的能力是重要的，例如，对某些感染、自身免疫和过敏性疾病的保护。然而，控制PKC独特功能和细胞内定位的机制是什么？在T淋巴细胞中(PKC家族的其他相关成员没有共享)，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结合。对这些途径的理解可能会为PKC的使用建立更合理的基础？作为自身免疫和过敏性疾病以及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