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

项目摘要 而丝氨酸、苏氨酸和酪氨酸的磷酸化是非常好的特征，相对 对组氨酸(His)的磷酸化知之甚少，它可能占所有参入的~6% 将磷酸盐转化为哺乳动物蛋白质。我们已经提供了遗传和生化证据证明组氨酸 激酶，NDPK-B，和组氨酸磷酸酶(PTase)，PHPT1和(PGAM5)，调节着 钙离子通过可逆性His磷酸化激活K+通道KCa3.1，从而激活CD4T和KCa3.1。 肥大细胞。NDPK-B His磷酸化激活KCa3.1，PGAM5和PHPT1抑制KCa3.1 His 通过特异性地去磷酸化和抑制NDPK-B和KCa3.1来实现磷酸化。vbl.使用 最近开发的抗1-和3-磷酸组氨酸(PHI)的单抗，我们首次证明 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。 SA1我们将在这些研究的基础上，评估他对NDPK-B和KCa3.1的磷酸化在 TCR信号转导的背景，决定它如何被各种信号分子调节，如PI3KC2，是否 其他PHI蛋白存在于T细胞中和/或受TCR信号调节，以及PGAM5的特定作用， PHPT1和NDPK-B调节PHIS蛋白的变化。我们发现24裂解了PGAM5-L 异构体对NDPK-B的脱磷最为关键。我们将确定24是否切割PGAM5-L 异构体对体内CD4T细胞的负性调节，TCR后这种异构体的数量是否发生变化 刺激，以及介导CD4T细胞分裂的膜内蛋白酶。 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学特性 亚基和KATP通道-/-小鼠。PHPT1-/-β细胞的缺陷可以用KATP通道的故障来解释 将细胞内隔室重新定位到质膜(PM)以响应低糖和 Leptin和我们现在已经将PHPT1-/-β细胞中的缺陷与瞬时受体电位的激活受损联系起来 频道4(TRPC4)。我们的假设是PHPT1，NDPK-B， PGAM5调节TRPC4通道活性的方式与KCa3.1相似，只是方向相反； 他对KCa3.1的磷酸化被激活，对TRPC4的磷酸化被抑制。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，它们的作用 在KATP通道交易和TRPC4激活中，以及在TRPC4中是否减少了对PM的KATP交易-/- 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 和志在一起。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 分子与人类疾病有关，包括CHI和2型糖尿病。

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.