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

项目总结 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则非常好。 特征是，对组氨酸的磷酸化知之甚少，这可能解释了 ~6%的磷酸盐掺入哺乳动物蛋白质中。蛋白激酶和磷酸酶的同一性 调节组氨酸磷酸化、它们的蛋白靶标和它们的生物学功能 默默无闻。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1(PHPT1) 调节钙激活的K+通道KCa3.1的活性，从而激活CD4T细胞和 桅杆。这些发现可能只是“冰山一角”，而是未来的基础 这是因为许多其他途径很可能是由组氨酸磷酸化调节的。我们现在有了 只鉴定了第二种组氨酸磷酸酶，它能特异性地使组氨酸去磷酸化并抑制 NDPK-B，并通过这种作用，是一种强大的负调节KCa3.1和CD4T细胞。此外，使用最近的 开发了抗1-和3-磷酸组氨酸(PHIs)的单抗，我们首次展示了 哺乳动物细胞体内组氨酸磷酸化的调节，这反过来又与TCR信号联系在一起。 我们的提案将解决这种新发现的组氨酸的调节、蛋白质靶标和生物学作用。 磷酸酶，这将扩展到一般性质的组氨酸磷酸化的调节 哺乳动物细胞中的生物过程。 在目标1中，我们将确定这种新发现的组氨酸磷酸盐的机制(S)。 调节并进而调节NDPK-B去磷酸化，调节下游通路的激活， 以及这种组氨酸磷酸酶作为组氨酸磷酸酶对其他PHIS蛋白的作用。此外，使用 最近开发的抗1-和3-PHIs抗体，结合其他产生的初步数据，我们提出了一种 识别新组氨酸磷酸化蛋白的新策略。在目标2中，我们将确定角色和 调节这种新发现的组氨酸磷酸酶以抑制免疫细胞信号。我们发现 小干扰RNA对人Th0和人外周血中Th0细胞组氨酸磷酸酶的抑制作用 基因敲除小鼠增加了NDPK-B的1-PHIs磷酸化，导致KCa3.1激活增加 随后TCR刺激的钙流通量和细胞因子的产生增加。我们将探索 TCR信号背景下这种磷酸酶的调节和功能(S)，它在特异性CD4T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。我们还将在TCR中扩展调查结果 CD4T细胞对FcεR1的信号刺激肥大细胞的激活。

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.