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.

项目摘要蛋白质的可逆磷酸化调节其功能，从而调节几乎所有的功能细胞过程。丝氨酸，苏氨酸和酪氨酸的磷酸化非常好特征是组氨酸的磷酸化相对较少，这可能是〜6％的磷酸掺入哺乳动物蛋白中。激酶和磷酸酶的身份调节组氨酸磷酸化，其蛋白质靶标及其生物功能一直保持朦胧。在过去的几年中，我们提供了组氨酸激酶，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，因此，CD4 T细胞和桅杆。这些发现可能仅代表“冰山一角”，并作为未来的基础工作是因为许多其他途径可能受组氨酸磷酸化调节。我们现在有仅鉴定出第二个组氨酸磷酸酶，该磷酸酶特异性组氨酸会脱磷酸化并抑制 NDPK-B，通过这种效果，是潜在的负调节剂KCA3.1和CD4 T细胞。而且，最近使用开发了对1-和3-磷酸二氨基丁烷（PHI）的单克隆抗体，我们首次证明调节哺乳动物细胞体内组氨酸磷酸化的调节，这又与TCR信号传导有关。我们的建议将解决该新确定的组氨酸的调节，蛋白质靶标和生物学作用磷酸酶，然后将其扩展到组氨酸磷酸化的一般特性哺乳动物细胞中的生物过程。在AIM 1中，我们将确定这种新鉴定的组氨酸磷酸盐的机制经过调节和调节NDPK-B的去磷酸化，调节下游途径的激活，以及这种组氨酸磷酸酶作为其他Phis蛋白的组氨酸磷酸酶的作用。另外，使用最近开发了抗1和3-Phis抗体，再加上生成的其他初步数据，我们提出了A 鉴定新的组氨酸磷酸化蛋白质的新型策略。在AIM 2中，我们将确定角色和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。我们发现在人Th0 CD4细胞和从从中分离的人Th0 CD4细胞和Th0 CD4细胞中组氨酸磷酸酶的siRNA敲低敲除小鼠的NDPK-B增加了1次磷酸化，导致KCA3.1的激活增加随后TCR刺激的Ca2+通量和细胞因子产生增加。我们将探索在TCR信号的背景下，该磷酸酶的调节和功能，其在特定CD4 T细胞中的作用子集，以及它在体内是否起作用以限制自身免疫性疾病。我们还将在TCR中扩展发现 CD4 T细胞中的信号传导对FcεR1刺激的肥大细胞激活。