PHOSPHOPROTEOMICS OF FGF-23 SIGNALING PATHWAY IN 1 ALPHA-HYDROXYLASE REGULATION

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 通过对 X 连锁低磷血症性佝偻病和肿瘤引起的骨软化症等罕见疾病的观察，FGF-23（一种由骨骼产生的循环激素）已成为磷和 1,25 二羟基维生素 D 代谢最重要的调节剂之一。体内和体外研究表明，FGF-23 通过抑制负责合成 1,25 二羟基维生素 D 的 1-α 羟化酶，抑制肾脏产生活性形式的维生素 D（1,25 二羟基维生素 D）。在之前的实验中，我们已经表明，通过 ERK1/2 激活 MAPK 信号通路对于诱导 FGF-23 在肾近曲小管上皮细胞中抑制 1-α 羟化酶是必要的。然而，FGF-23 调节 1-α 羟化酶表达的确切机制尚不清楚。 该项目的目的是利用定量磷酸蛋白质组学更好地了解肾近曲小管细胞中 FGF-23 激活的信号通路。我们将使用 SILAC（细胞培养物中氨基酸的稳定同位素标记）来比较经过和未经 FGF-23 处理的差异标记细胞群之间的磷蛋白水平。为了富集磷酸肽，我们将通过免疫沉淀酪氨酸磷酸化蛋白进行亲和纯化。磷酸肽的鉴定将由 UCSF 质谱设备使用混合质谱仪 (LTQ-Orbitap) 进行。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. From the observation of rare diseases like X-linked hypophosphatemic rickets and tumor-induced osteomalacia, FGF-23, a circulating hormone produced by bone has emerged as one of the most important regulators of phosphorus and 1,25 dihydroxyvitamin D metabolism. In vivo and in vitro studies have shown that FGF-23 inhibits the renal production of active form of vitamin D (1,25 dihydroxyvitamin D), by suppression of the 1-alpha hydroxylase enzyme which is responsible for the synthesis of 1,25 dihydroxyvitamin D. In previous experiments, we have shown that activation of MAPK signaling pathway via ERK1/2 is necessary to induce suppression of the 1-alpha hydroxylase enzyme by FGF-23 in renal proximal tubule epithelial cells. However, the exact mechanism of regulation of 1-alpha hydroxylase enzyme expression by FGF-23 is unknown. The aim of this project is to better understand the signaling pathway activated by FGF-23 in renal proximal tubule cells using quantitative phosphoproteomics. We will use SILAC (stable isotope labeling with amino acids in cell culture) to compare phosphoprotein levels between differentially labelled cell populations, with and without treatment with FGF-23. In order to enrich for phosphopeptides, we will perform affinity purification with immunoprecipitation of tyrosine phosphorylated proteins. Identification of phosphopeptides will be performed by the UCSF Mass Spectrometry Facility using a hybrid mass spectrometer (LTQ-Orbitap).