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.

项目摘要丝氨酸，苏氨酸和酪氨酸的磷酸化更为完全，相对关于组氨酸（HIS）的磷酸化知之甚少，这可能占所有收入的约6％磷酸盐成哺乳动物蛋白。我们提供了组氨酸的遗传和生化证据激酶，NDPK-B和组氨酸磷酸酶（PTases），PHPT1和（PGAM5）调节 Ca2+激活的K+通道KCA3.1通过可逆他的磷酸化，从而激活CD4 T和肥大细胞。而NDPK-B他的磷酸化并激活KCA3.1，PGAM5和PHPT1抑制KCA3.1他分别通过特异性去磷酸化和抑制NDPK-B和KCA3.1的磷酸化。使用最近开发了对1和3-磷酸 - 抗二氨酸（PHIS）的单克隆抗体，我们首次证明在哺乳动物细胞中体内组氨酸磷酸化的调节，我们又与TCR信号传导有关。 SA1我们将基于这些研究，我们将评估他在NDPK-B和KCA3.1磷酸化中的变化 TCR信号的上下文，确定如何通过各种信号分子（例如PI3KC2）调节其其他Phis蛋白存在于T细胞中，//或受TCR信号的调节，PGAM5的特定作用， PHPT1和NDPK-B调节PHIS蛋白的变化。我们发现24裂解PGAM5-L 同工型对于去磷酸化的NDPK-B最重要。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，该同工型的量是否在TCR后发生变化刺激以及介导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 来自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细胞中的缺陷可以通过KATP通道的失败来解释从葡萄糖和低葡萄糖和 Leptin，我们现在将PHPT1 - / - β细胞中的缺陷与瞬时受体电位的激活相关联频道4（TRPC4）。我们的假设是，由PHPT1，NDPK-B，NDPK-B，NDPK-B的磷酸化可逆 PGAM5以与KCA3.1相似的方式调节TRPC4通道活性，尽管朝相反的方向调节。然而他的KCA3.1磷酸化激活，他的TRPC4磷酸化抑制。在SA 2中，我们将确定是否 PHPT1，NDPK-B和PGAM5以类似于KCA3.1的方式调节TRPC4的磷酸化在KATP渠道贩运和TRPC4激活中，以及在Trpc4中的KATP贩运降低到PM PGAM5 - / - 小鼠牵引先天性高胰岛素血症低血糖，与PHPT - / - 小鼠和患者相似与Chi。然后，我们将将这些研究扩展到人类细胞，并评估这些研究的潜在相关性包括CHI和2型糖尿病的人类疾病分子。