Identification and characterization of a novel mammalian histidine phosphatase that negatively regulates CD4 T cells

负调节 CD4 T 细胞的新型哺乳动物组氨酸磷酸酶的鉴定和表征

• 批准号: 9330534
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 54.79万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-19 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330534
• 关键词: Accounting; Address; Antibodies; Autoimmune Diseases; Autoimmunity; Binding; Biochemical; Biological; Biological Process; Biology; CD4 Positive T Lymphocytes; Calcium-Activated Potassium Channel; Cell physiology; Cells; Coupled; Cytosol; Data; Disease; Enzymes; Exhibits; Family; Foundations; Future; Genetic; Genetic Transcription; Goals; Growth and Development function; Histidine; Human; Hypersensitivity; Immune; Knockout Mice; Link; Mammalian Cell; Mediating; Membrane Potentials; Mitochondria; Modeling; Modification; Monitor; Monoclonal Antibodies; Mus; Pathway interactions; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Process; Production; Property; Protein Dephosphorylation; Protein Isoforms; Protein Tyrosine Phosphatase; Protein phosphatase; Proteins; Reagent; Receptor Activation; Receptor Signaling; Regulation; Reporting; Role; Serine/Threonine Phosphorylation; Signal Pathway; Signal Transduction; Small Interfering RNA; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Time; Tyrosine; Work; cell growth; cytokine; graft vs host disease; immune activation; in vivo; inorganic phosphate; insight; knock-down; mast cell; novel; novel strategies; nucleoside diphosphate; protein-histidine kinase; response

Project summary Reversible phosphorylation of proteins modulates their function and thereby regulates virtually all cellular processes. Whereas phosphorylation of serine, threonine and tyrosine are exceedingly well characterized, relatively little is known about phosphorylation of histidine, which may account for as much as ~6% of all incorporation of phosphate into mammalian proteins. The identity of the kinases and phosphatases that regulate histidine phosphorylation, their protein targets, and their biological functions have remained obscure. Over the past several years, we provided genetic and biochemical evidence that the histidine kinase, nucleoside diphosphate-B (NDPK-B), and the histidine phosphatase, protein histidine phosphatase 1 (PHPT1) regulate the activity of the Ca2+-activated K+ channel KCa3.1 and, therefore the activation of CD4 T cells and mast. These discoveries likely represent only the “tip of the iceberg” and serve as the foundation for future work because it is likely that many other pathways are regulated by histidine phosphorylation. We have now identified only the second histidine phosphatase, which specifically histidine dephosphorylates and inhibits NDPK-B and, via this effect, is a potent negative regulator KCa3.1 and CD4 T cells. Moreover, using recently developed monoclonal antibodies to 1- and 3-phospho-Histidine (pHis), we demonstrate for the first time the regulation of histidine phosphorylation in vivo in mammalian cells, which we in turn linked to TCR signaling. Our proposal will address the regulation, protein targets, and biologic roles for this newly identified histidine phosphatase, which will then be extended to general properties of histidine phosphorylation in regulating biological processes in mammalian cells. In Aim 1, we will identify the mechanism(s) whereby this newly identified histidine phosphates is regulated and in turn regulates NDPK-B dephosphorylation, modulates activation of downstream pathways, and the role of this histidine phosphatase as a histidine phosphatase for other pHis proteins. In addition, using recently developed anti-1- and 3-pHis antibodies, coupled with other preliminary data generated, we propose a novel strategy to identify new histidine phosphorylated proteins. In Aim 2, we will determine the role and regulation of this newly identified histidine phosphatase to suppress immune cell signaling. We found that siRNA knockdown of the histidine phosphatase in human Th0 CD4 cells and Th0 CD4 cells isolated from knockout mice have increased 1-pHis phosphorylation of NDPK-B, leading to increased activation of KCa3.1 and a subsequent increase in TCR-stimulated Ca2+ flux and cytokine production. We will explore the regulation and function(s) of this phosphatase in the context of TCR signaling, its role in specific CD4 T cell subsets, and whether it functions in vivo to limit autoimmune disease. We will also will extend findings in TCR signaling in CD4 T cells to FcεR1-stimulated activation of mast cells.

项目摘要 蛋白质的可逆磷酸化调节它们的功能，从而调节几乎所有 细胞过程而丝氨酸、苏氨酸和酪氨酸的磷酸化非常好， 特征，相对较少了解组氨酸的磷酸化，这可能占多达 ~6%的磷酸盐掺入哺乳动物蛋白质中。激酶和磷酸酶的鉴别 调节组氨酸磷酸化的蛋白质，它们的蛋白质靶点和它们的生物学功能 晦涩难懂。在过去的几年里，我们提供了遗传和生化证据， 核苷二磷酸-B（NDPK-B）和组氨酸磷酸酶，蛋白组氨酸磷酸酶1（PHPT 1） 调节Ca 2+激活的K+通道KCa3.1的活性，从而激活CD 4 T细胞， 桅杆这些发现可能只是“冰山一角”，并作为未来的基础。 这是因为许多其他途径可能受组氨酸磷酸化调节。我们现在已经 仅鉴定了第二种组氨酸磷酸酶，其特异性地使组氨酸去磷酸化并抑制 NDPK-B，通过这种作用，是一种有效的负调节KCa3.1和CD 4 T细胞。此外，最近使用 开发的单克隆抗体1-和3-磷酸组氨酸（pHis），我们首次证明， 在哺乳动物细胞中，组氨酸磷酸化的体内调节，我们又将其与TCR信号传导联系起来。 我们的建议将解决这个新发现的组氨酸的调节，蛋白质靶点和生物学作用 磷酸酶，然后将扩展到组氨酸磷酸化的一般性质，在调节 哺乳动物细胞中的生物过程。 在目标1中，我们将确定这种新鉴定的组氨酸磷酸盐的作用机制。 调节并反过来调节NDPK-B去磷酸化，调节下游途径的激活， 以及这种组氨酸磷酸酶作为其它pHis蛋白的组氨酸磷酸酶的作用。此外，使用 最近开发的抗1-和3-pHis抗体，加上其他初步数据产生，我们提出了一个 新的策略，以确定新的组氨酸磷酸化蛋白质。在目标2中，我们将确定角色， 调节这种新鉴定的组氨酸磷酸酶以抑制免疫细胞信号传导。我们发现 siRNA敲低人Th 0 CD 4细胞和分离自 敲除小鼠NDPK-B的1-pHis磷酸化增加，导致KCa3.1活化增加 以及随后TCR刺激的Ca 2+通量和细胞因子产生的增加。我们将探讨 在TCR信号传导的背景下，该磷酸酶的调节和功能，其在特异性CD 4 T细胞中的作用， 亚群，以及它是否在体内发挥作用以限制自身免疫性疾病。我们还将扩大TCR的调查结果， CD 4 T细胞中的信号传导至FcεR1刺激的肥大细胞活化。