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

项目摘要 尽管丝氨酸、苏氨酸和酪氨酸的磷酸化非常好地表征，相对而言， 组氨酸（His）的磷酸化可能占所有掺入的约6%，但对它的了解很少 磷酸盐转化成哺乳动物蛋白质。我们已经提供了遗传和生化证据， 激酶NDPK-B和组氨酸磷酸酶（PTases）PHPT 1和（PGAM 5）调节细胞的活性。 Ca2+通过可逆的His磷酸化激活K+通道KCa3.1，从而激活CD4 T细胞， 肥大细胞NDPK-B His磷酸化激活KCa 3.1，而PGAM 5和PHPT 1抑制KCa 3.1 His 通过特异性地去磷酸化和抑制NDPK-B和KCa 3.1来抑制磷酸化。使用 最近开发的单克隆抗体1-和3-磷酸组氨酸（pHis），我们首次证明 哺乳动物细胞中组氨酸磷酸化的体内调节，我们又将其与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KCa 3.1的His磷酸化的变化， TCR信号传导的背景下，确定它是如何调节各种信号分子，如PI3KC2 β，是否 其它pHis蛋白存在于T细胞中和/或受TCR信号传导调节，而PGAM5， PHPT1和NDPK-B调节pHis蛋白的变化。我们已经发现，PGAM5-L的124切割 同种型对NDPK-B去磷酸化最关键。我们将确定PGE24是否切割PGAM5-L 同种型在体内负调节CD4 T细胞，无论该同种型的量在TCR后是否改变， 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癌细胞功能中的关键作用。我们发现 来自PHPT 1-/-小鼠的EPCs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。PHPT 1-/-β细胞的这种缺陷可以通过KATP通道的失效来解释 响应于低葡萄糖从细胞内区室重新定位到质膜（PM）， 我们现在已经将PHPT1-/-β细胞中的缺陷与瞬时受体电位激活受损联系起来 通道4（TRPC 4）。我们的假设是，TRPC 4的可逆His磷酸化通过PHPT 1，NDPK-B， PGAM 5以与KCa 3.1类似的方式调节TRPC 4通道活性，尽管方向相反; His对KCa3.1的磷酸化激活，His对TRPC 4的磷酸化抑制。在SA 2中，我们将确定 PHPT 1、NDPK-B和PGAM 5以类似于KCa 3.1的方式调节TRPC 4的His磷酸化，它们的作用 在KATP通道运输和TRPC 4激活中，以及在TRPC 4-/- PGAM5-/-小鼠导致与PHPT-/-小鼠和患者相似的先天性高胰岛素血症低血糖 在CHI。然后，我们将把这些研究扩展到人类胰岛细胞，并评估这些研究的潜在相关性。 分子对人类疾病包括CHI和2型糖尿病的作用。

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.