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Histidine Phosphorylation in Mammals: Regulation, Protein Targets, and Biology

哺乳动物中的组氨酸磷酸化：调节、蛋白质靶点和生物学

基本信息

批准号：
10152661
负责人：
EDWARD Y SKOLNIK
金额：
$ 39.83万
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：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10152661
关键词：
Adenocarcinoma Autoimmune Diseases B-Lymphocytes Beta Cell Binding Biochemical Biological Biological Process Biology CD4 Positive T Lymphocytes Calcium-Activated Potassium Channel Cell membrane Cell physiology Cells Cleaved cell Defect Diabetes Mellitus Disease Exhibits Failure Family Genetic Glucose Hepatic Histidine Human Hypersensitivity Hypoglycemia Immune Impairment Internships Leptin Link Mammalian Cell Mammals Mediating Metabolic Monoclonal Antibodies Mus Mutation Neonatal Hypoglycemia Newborn Infant Non-Insulin-Dependent Diabetes Mellitus Pancreas Pathway interactions Patients Peptide Hydrolases Persistent Hyperinsulinemia Hypoglycemia of Infancy Phenotype Phosphoglycerate Mutase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Play Potassium Process Protein Dephosphorylation Protein Isoforms Proteins Protomer Reagent Receptor Activation Receptor Signaling Regulation Role Second Messenger Systems Serine Signal Pathway Signal Transduction Signaling Molecule Structure of beta Cell of islet T-Cell Activation T-Cell Receptor T-Lymphocyte TRP channel Threonine Time Tumor Immunity Tumor Suppressor Proteins Tyrosine complement C2b electrical property human disease in vivo inorganic phosphate insight mast cell nucleoside diphosphate phosphohistidine protein-histidine kinase response trafficking

项目摘要

Project Summary Whereas phosphorylation of serine, threonine and tyrosine are exceedingly well characterized, relatively little is known about phosphorylation of histidine (His), which may account for as much as ~6% of all incorporation of phosphate into mammalian proteins. We have provided genetic and biochemical evidence that the histidine kinase, NDPK-B, and the histidine phosphatases (PTases), PHPT1 and (PGAM5, regulate the activity of the Ca2+-activated K+ channel KCa3.1 by reversible His phosphorylation, and thereby the activation of CD4 T and mast cells. While NDPK-B His phosphorylates and activates KCa3.1, PGAM5 and PHPT1 inhibit KCa3.1 His phosphorylation by specifically dephosphorylating and inhibiting NDPK-B and KCa3.1 respectively. 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. SA1 we will build on these studies, we will assess changes in His phosphorylation of NDPK-B and KCa3.1 in the context of TCR signaling, determine how it is regulated by various signaling molecules such as PI3KC2, whether other pHis proteins are present in T cells and/or regulated by TCR signaling, and the specific role for PGAM5, PHPT1 and NDPK-B to modulate changes in pHis proteins. We have found that the 24 cleaved PGAM5-L isoform is most critical to dephosphorylate NDPK-B. We will determine whether the 24 cleaved PGAM5-L isoform negatively regulates CD4 T cells in vivo, whether the amount of this isoform changes following TCR stimulation, and the intramembranous proteases that mediates cleavage in CD4 T cells. We also identified a critical role for histidine phosphorylation in pancreatic  cell function. We found that  cells from PHPT1-/- mice have electrical properties similar to those of patients with mutations in KATP channel subunits and KATP channel-/- mice. The defect in PHPT1-/- β cells can be explained by the failure of KATP channels to relocalize from an intracellular compartment to the plasma membrane (PM) in response to low glucose and leptin and we have now linked the defect in PHPT1-/- β cells to impaired activation of transient receptor potential channel 4 (TRPC4). Our hypothesis is that reversible His phosphorylation of TRPC4 by PHPT1, NDPK-B, PGAM5 regulates TRPC4 channel activity in a similar manner to KCa3.1, albeit in opposite directions; whereas His phosphorylation of KCa3.1 activates, His phosphorylation of TRPC4 inhibits. In SA 2, we will determine if PHPT1, NDPK-B, and PGAM5 regulate His phosphorylation of TRPC4 in a manner similar to KCa3.1, their role in KATP channel trafficking and TRPC4 activation, and whether decreased KATP trafficking to the PM in TRPC4-/- and PGAM5-/- mice leads congenital hyperinsulinemia hypoglycemia that is similar to PHPT-/- mice and patients with CHI. We will then extend these studies to human  cells and assess the potential relevance of these molecules to human disease that include CHI and type 2 diabetes mellitus.

项目概要尽管丝氨酸、苏氨酸和酪氨酸的磷酸化已得到非常充分的表征，但相对而言关于组氨酸 (His) 的磷酸化知之甚少，组氨酸 (His) 的磷酸化可能占所有掺入量的约 6% 将磷酸盐转化为哺乳动物蛋白质。我们提供了遗传和生化证据表明组氨酸激酶、NDPK-B 和组氨酸磷酸酶 (PTase)、PHPT1 和 (PGAM5) 调节 Ca2+ 通过可逆的 His 磷酸化激活 K+ 通道 KCa3.1，从而激活 CD4 T 和肥大细胞。 NDPK-B His 磷酸化并激活 KCa3.1，而 PGAM5 和 PHPT1 抑制 KCa3.1 His 分别通过特异性去磷酸化和抑制 NDPK-B 和 KCa3.1 来实现磷酸化。使用我们首次展示了最近开发的针对 1- 和 3-磷酸-组氨酸 (pHis) 的单克隆抗体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。 SA1，我们将在这些研究的基础上，我们将评估 NDPK-B 和 KCa3.1 磷酸化的变化 TCR 信号传导的背景，确定它如何受到各种信号分子（例如 PI3KC2）的调节，是否其他 pHis 蛋白存在于 T 细胞中和/或受 TCR 信号传导调节，以及 PGAM5 的具体作用， PHPT1 和 NDPK-B 调节 pHis 蛋白的变化。我们发现 24 裂解的 PGAM5-L 同种型对于 NDPK-B 去磷酸化最为关键。我们将确定 24 是否裂解 PGAM5-L 同工型在体内负调节 CD4 T 细胞，TCR 后该同工型的数量是否发生变化刺激和介导 CD4 T 细胞裂解的膜内蛋白酶。我们还确定了组氨酸磷酸化在胰腺  细胞功能中的关键作用。我们发现 PHPT1-/- 小鼠的  细胞具有与 KATP 通道突变患者相似的电特性亚基和 KATP 通道-/- 小鼠。 PHPT1-/- β 细胞的缺陷可以通过 KATP 通道的故障来解释响应低葡萄糖和从细胞内区室重新定位到质膜（PM）瘦素，我们现在已经将 PHPT1-/- β 细胞的缺陷与瞬时受体电位激活受损联系起来通道 4 (TRPC4)。我们的假设是 PHPT1、NDPK-B 对 TRPC4 的 His 磷酸化是可逆的， PGAM5 以与 KCa3.1 类似的方式调节 TRPC4 通道活性，尽管方向相反；然而 His 磷酸化 KCa3.1 激活，His 磷酸化 TRPC4 抑制。在 SA 2 中，我们将确定是否 PHPT1、NDPK-B 和 PGAM5 以与 KCa3.1 类似的方式调节 TRPC4 的 His 磷酸化，它们的作用 KATP 通道运输和 TRPC4 激活的影响，以及 TRPC4-/- 中 KATP 向 PM 的运输是否减少和 PGAM5-/- 小鼠导致先天性高胰岛素血症低血糖，与 PHPT-/- 小鼠和患者相似与气。然后，我们将把这些研究扩展到人类  细胞，并评估这些研究的潜在相关性。人类疾病的分子，包括 CHI 和 2 型糖尿病。