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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要蛋白质的可逆磷酸化调节其功能并调节几乎所有细胞过程，包括丝氨酸、苏氨酸和酪氨酸的磷酸化都非常好。特征，关于组氨酸的磷酸化知之甚少，这可能解释了约 6% 的磷酸盐掺入哺乳动物蛋白质中激酶和磷酸酶的特性。调节组氨酸磷酸化、其蛋白质靶标及其生物学功能仍然存在在过去的几年里，我们提供了组氨酸激酶的遗传和生化证据，核苷二磷酸-B (NDPK-B) 和组氨酸磷酸酶、蛋白组氨酸磷酸酶 1 (PHPT1) 调节 Ca2+ 激活的 K+ 通道 KCa3.1 的活性，从而调节 CD4 T 细胞的激活这些发现可能仅代表“冰山一角”，并作为未来的基础。之所以起作用，是因为我们现在已经掌握了许多其他途径可能受到组氨酸磷酸化的调节。仅鉴定了第二种组氨酸磷酸酶，该酶特异性地使组氨酸去磷酸化并抑制 NDPK-B 通过这种作用，成为 KCa3.1 和 CD4 T 细胞的有效负调节剂。开发了针对 1- 和 3-磷酸-组氨酸 (pHis) 的单克隆抗体，我们首次证明了哺乳动物细胞体内组氨酸磷酸化的调节，我们进而将其与 TCR 信号传导联系起来。我们的提案将解决这种新发现的组氨酸的调控、蛋白质靶标和生物学作用磷酸酶，然后将其扩展到调节组氨酸磷酸化的一般特性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，以及该组氨酸磷酸酶作为其他 pHis 蛋白的组氨酸磷酸酶的作用。最近开发的抗 1- 和 3-pHis 抗体，再加上生成的其他初步数据，我们提出了识别新组氨酸磷酸化蛋白的新策略在目标 2 中，我们将确定其作用和作用。我们发现，这种新发现的组氨酸磷酸酶可以抑制免疫细胞信号传导。 siRNA 敲低人 Th0 CD4 细胞和分离自 Th0 CD4 细胞的组氨酸磷酸酶敲除小鼠 NDPK-B 的 1-pHis 磷酸化增加，导致 KCa3.1 的激活增加以及随后 TCR 刺激的 Ca2+ 通量和细胞因子产生的增加。该磷酸酶在 TCR 信号传导背景下的调节和功能，及其在特定 CD4 T 细胞中的作用子集，以及它是否在体内发挥作用以限制自身免疫性疾病，我们还将扩展 TCR 的发现。 CD4 T 细胞中的信号传导至 FcεR1 刺激的肥大细胞激活。